CA2132949A1 - Stability-enhanced variants of parathyroid hormone - Google Patents
Stability-enhanced variants of parathyroid hormoneInfo
- Publication number
- CA2132949A1 CA2132949A1 CA002132949A CA2132949A CA2132949A1 CA 2132949 A1 CA2132949 A1 CA 2132949A1 CA 002132949 A CA002132949 A CA 002132949A CA 2132949 A CA2132949 A CA 2132949A CA 2132949 A1 CA2132949 A1 CA 2132949A1
- Authority
- CA
- Canada
- Prior art keywords
- pth
- stability
- variant
- variant according
- enhanced
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Herein described are variants of parathyroid hormone that retain significant PTH activity and are substantially resistant to trypsin and trypsin-like enzymes. The variants are useful pharmaceutically, to treat bone disorders such as osteoporosis and in other therapeutic applications. Specific embodiments of the invention include [His25]PTH, [His26G1n27]PTH and [His25His26Leu27]PTH.
Description
WO 93J20203 PCl`/CA93/00136 8TA~ Y--IZ~Cl!:D VA~IAN'r~ OF PA~TIIYROID ~O~MONE ::
Field of ~he Invention ~-This invention relates to variants of parathyroid hormone, to the produetion of such variant~ particularly S via reeombinant DNA teehnology and to the use of such ~
variants as therapeutie agents, for example in the ~ :
treatment of oQteoporosis.
Baekaround to the Invention Parathyroid hormone (PTH) is a seereted, protein produet of ma~malian parathyroid glands that regulates ealeium ho~eostasis through its aetion on various tissues, ineluding bone and vaseular tissue. Reaeareh into the physiologieal role o~ PTH has identified elinieally relevant effeets on bone metabolism, there being. so~e elinieal evidenee that PTH may be useful in the treat~ent of ost~oporo~is and related o~teopenie a~flietions. An effeet on vaseular tissue and on keratinoeyte growth has also been noted.
To obtain PTH in the a~ounts required for elinieal investigations and for eommereial purposQs, reeombinant DNA-based teehnique~ have been sueeessfully applied in -~
the produetion of sueh mammalian PTH speeies as human ;~
PTH, bovine PTH, poreine PTH and rat PTH, whieh in their mature form all eontain 84 amino aeids arranged in a speeies-speeifie sequenee. An understanding of the strueture of these proteins, and partieularly human PTH --and bovine PTH, has lead al~o to the diseovery that PTH
aetivity ean be attributed to the first 34 N-terminal residues of the mature hormone. This ha~ allowed for the production of biologically active PTH fragments using the ~olid phase technique of peptide synthe~is, to meet PTH ~-demand.
In the interest of furthering development of PTH as a pharmaceu~ical product, it would be desirable to provide stability-enhanced forms of PTH that are better ~uited for hu~n administration. It is known from studies with serum-derived PTH samples, and from ' " "..
,, " ..
:
~ ~ j~J !~
experience with recombinant PTH production, that the ;
hormone is particularly vulnerable to protease digestion. ~:
PTH production in yeast, for example, has shown that PTH
is degraded by the yeast-produced kex enzyme, which -recognizes dibasic residues. When the lysine residue at po~ition 26 of PTH is replaced with glutamine, however, -~
proteolytic degradation of PTH by the yeast is reportedly reduced (~ee Reppe et al, J. Biol. Chem., 1991, 266:14198). Other studie~ targetting thi~ general region o~ the PTH molecule have indicated that amino acid replacement can cause significant decline in PTH ;~
activity. Conversion of the ly~ine at position 27 for exa~pl~, resulted in a marked decline in PTH activity (see Wingender et al, WO 90/10067) as did derivatization lS of the arginine residue~ at positions 25 and 20 (see Rosenblatt et al, 1978, Biochemistry, 17:3188).
It is an object of the present invention to provide a novel variant of parathyroid hormone.
It is another object of the present invention to 20 provide a parathyroid hormone variant that exhibits -improved stability in the presence of proteolytic enzy~es, e~pecially trypsin and trypsin-like enzymes.
It is another object of the present invention to ~-provide a parathyroid hormone variant that exhibits both 25 improved stability in the presen e of proteolytic enzymes -~
and a bioactivity that is comparable to parathyroid hormone.
It is another object of the preaent invention to `~
prov~de a pharmaceutical composition comprisinq a parathyroid hormone variant exhibiting improved stability and comparable activity, for use in therapeutic applications.
It is another object of the present invention to provid~ a process for producing a stability-enhanced -variant of parathyroid hormone.
5~= '~ ' ' : ` ~
WOg3/20203 PCT/CA93/00136 ~, ,' 3 '~' The present invention provides variants of ~-' parathyroid hormone that are altered structurally to confer enhanced stability in the presence of proteolytic '~-enzymes such as trypsin and the so-called trypsin-like S enzymes that cleave at internal arginine and lysine re~idues. Although numerous putative tryp~in cleavage ', sites reside in native parathyroid hormone, it has surprisingly been found that substantial resistance to tryptic digestion is conferred when the hormone is 10 altered within a single region con~titut-d by re~idues ~-, Arg~Lys~Ly~n. It has further been found that variants -having activity comparable to native parathyroid hormone ''~
are generated whQn replacemQnt amino acids are se,lected appropriately. Thus, the PTH variants of the present lS invention are well suited for therapeutic applications ~-~
and are b~tter adapted to survive expo~ure particularly to tryp~in and trypsin-like enzy~es while in the p~ripheral circulation and al~o during their production, , handling and storage. ;~
~ccording to one aspect of the pre~ent invention, there is provided a stability-enhanced variant of a parathyroid hor~one co~pound harbouring the region -~
Arg25Lys~Lysn, in which said region is replaced in said variant by an amino acid sequence selected fr~m~
(a) X~Lys26Lys~; -(b) Arg25y26z~ "
(c~ X2SY~Lys~; , (d) X~Lys~Z~; and '`"~
(e) XUY~Z~
wherein X, Y and Z are independently selectad trypsin-insensitive amino acids i.e. amino acids other than lysine and arginine. - '-In a preferred embodimen~ of the invention, the ' ~, replacement a~ino acids X, Y and Z are selected to yield ' ','-3S variant~ o~ hu~n PTH that in addition to exhibiting enh~nced stability in the presence of tryp~in, al~o '~,~
exhibit an activity comparable to native human PTH. In '~' ;, :' .
W093/20203 ~ ~S~t~ Y PCT/CA93/00136 :s specific embodiments of the invention, the replacement amino acids are selected from among the genetically encoded amino acids, to permit production of the variants -via recombinant DNA-based techniques.
According to another aspect of the pre~ent invention, there is provided a pharmaceutical composition, comprising a therapeutically effective amount of a parathyroid hormone variant of the present invention and a pharmaceutically acceptable carrier.
According to another aspect of the present invention, there is provided a method for treating a ma~oal, which comprises the step of administering to a ~a~oal in need thereof a pharmaceutical composition lS co~pri~ing a therap~utically effective amount of a parathyroid hormone variant of the present invention and a phar~aceutically acceptable carrier.
According to another aspect of the present invention, there is provided a process for producing a ~ ~-parathyroid hormone variant that exhibits enhanced ~tability in ~he presence of trypsin, which comp~ises the step of culturing a cellular host having incorporated expressibly therein a DNA molecule which codes for a PTH - ~-variant consisting only of genetically encoded amino 25 acids. -The invention and its preferred embodiments are now described in greater detail with reference to the accompanying drawings, in which:
Brief Reference to the Drawinas Figure l illustrates a recombinant DNA plas~id harbouring human PTH-encoding DNA, for expression in an E. coli host; and Figure 2 illustrates the sequence of the human PTH-encoding DNA incorporated on the plas~id of Figure 1, and 3S provides the sequence of human PTH with the Arg~Lys~Lys~
region identified by boxing.
Detailed Descri~tiOn of the Invention and Prefer~d Embodiments The invention relates to parathyroid hormone ;~
variants that exhibit improved stability in the presence ~
5 of trypsin and, in accordance with a preferred aspect of ~ -the invention, also exhibit activity at least similar to native PTH. In the pre~ent ~pecification, PTH activity is defined in the context o~ the o~teo~arcoma-based adenylate cycla~e a~ay employed conventionally in the art. Briefly, this a~say provide~ an in vitro determination o~ tbe extent to hich PTH stimulates adenylate cyclase activity in rat osteosarcoma cells of the `UMR' lineage, and thus provides an indication of PTH
effects on bone ti~ue in vi~o. Protocols for conducting the a~ay have been de5cribed by Rodan et al, 1983, J. -Clin. Inve~t., 72:15~1 (in which the osteo~aracoma cells of the ROS lineage are employed) and by Rabb~ni at al, - --1988, Endocrinol., 123:2?09 (which employ~ the line UMR~
106). PTH variants that exhibi~, in the UMR-ba~ed assay, an EC~ of at least 1,000 nM i.e. 1,000 nM or lower, are --herein characterized as having activity Usimilar~ to native PTH; a variant having an EC~ of lOOnM or lower is characterized as having activity Usubstantially similar"
to native PTH; and a variant having an EC~ of 20nM and lower is characterized as having an activity that is "comparable" to native PTH. The term EC~ refers to the concentration of PTH or variant effective for half-maximal stimulation of adenylate cyclase activity in the UMR-ba~ed as~ay.
PTH variants having an "improved stability in the presence of trypsinH are degraded ~y trypsin a~ a rate that is slower than a similarly treated native PTH
control. An assay suitable for identifying a reduced tryptic digestion rate entails a two step procedure, in 3S which a PTH variant and a native P~H control are ~-~eparatQly incubated witb trypsin for a de~ined period, ~-and are then assayed for activity in tbe osteosarcoma~
~':''.'~ ....
W093/20203 ~ j PCT/CA93/00136 based assay just described Protocols suitable for assaying trypsin sensitivity are described in the literature and are outlined in the examples herein In ~;~
this assay, variants of human PTH, for example, will exhibit an activity that is at least greater than a similarly treated native human PTH control In a quantitative context, the PTH variants ~ay be characterized ac having an improved stability in the -pr-sence of trypsin if the variant exhibits an EC~ as determined in the UMR-based adenylate cyclase a~say that is at least lower than a similarly treated native PTH
counterpast Under conditions of the assay exemplified her~in, for instance, native human PTH xhibits an EC~ of about 20nM or greater following trypsin incubation ~Stab~lity improved" human PTH variants are accordingly characterized by an EC~ of lower than 20nM, when assayed under these particular conditions In the present specification, a~ino acids are a~igned numerals to identify their location relative to the N-ter~inal amino acid of mature PTH For consistency and as is conventional in the art, amino acids are assigned the same positional number when present in the context of N-terminally truncated or extended forms of PTH.
In accordance with the present invention, PTH --variant~ exhibiting improved stability in the presence of trypsin are obtained when the Arg~Lys~Lysn region of PTH
i5 replaced with an amino acid sequence selected from (a) X2SLys~Lys27;
(b) Arg25y~zn (c) X25y2~LySn;
(d) X75Lys~Z~; and (e) X2sy26zn wherein X, Y and Z are independently selected, tryp~in-in~en~itive a~ino acids, i e a~ino acids other th~n ~rginine and lysine wo 93/20203 ~ 9 PCT/CA93/OOt36 It has surprisingly been found that measurably improved stability in the presence of trypsin is realized when the Arg25Lys26Lys27 region is altered by replacement merely of the Arg25 residue. Thus, according to one embodiment of the present invention, there is provided a PTH variant having improved stability in the presence of ~;
trypsin, in which Arg25 is replaced by an amino acid other than arginine and ly~ine.
According to another embodimsnt of tbs present invention, PTH variants exhibiting improved stability in the presence of tryp~in are obtained by replacing both ~ -Arg~ and Lys26 with independently selected amino acids ;
other than arginine and lysine. -According to another embodiment of the present -~
IS invsntion, PTH variants exhibiting improved stability in the pressnce of trypsin are obtained by replacing both .
the Arg25 and Lys~ with independently selected amino acids other than arginine and lysine.
According to another embodiment of the present ~ ~
20 invention, PTH variants exhibiting improved stability in i --the pre~ence of trypsin are obtained by replacing both ~-the Lys26 and Lysn with independently selected a~ino acids other than arginine and lysine.
PTH variants in which the entire Arg~Lys26Lys27 region 25 is replaced have been found to exhibit substantial ~ -resistance to tryptic digestion. According to a preferred ~-embodiment of the present invention therefore, the PTH
variants of the present invention are obtained by replacing each of the a~ino acids Arg25Lys26Lysn with an 30 independently selected amino acid other than arginine and ~ ~-lysine.
The replacement amino acids X, Y and Z in the above ~ -formulae are trypsin-insensitive amino acids and may be ~-~
cho~en from among the various ~ynthetic amino acids and ~ -3S th~ naturally occurring amino acids other than L-arginine ~nd L-ly~ine. ~n a preferred a~pect o~ the pre~ent ~
invention, the replace~ent amino acids X, Y and Z are ~ -.- ~
WOg3/20~03 ~ ~ PCT/CA93/00136 selected with a view to generating variants that exhibit activity comparable to native PTH. For this purpose, the ~-replacement amino acids are selected from among those amino acids which preserve the same configuration in this region of the PT~ molecule. More particularly, t~e replacement amino acids are most desirably selected from among those amino acids having either neutral or positively charged amino acid side-chains. UQeful amino acid replacements having neutral side chains include glycine, alanine, valine, leucine, isoleucine, ~erine, threonine, a~paragine, glutamine, phenylalanine, cysteine, tryptophan, tyrosine, methionine, proline, as well as the ~ynthetic analo~ues thereof, such a~
norleucine, norvaline, cyclohexylalanine, etc. Amino acids h~ving positively charged side chaina include hi~tidine and synthetic histidine analogue~, such as D-hi~tidine, l-methyl-L-histidine, 3-methyl-L-hist~dine, N-imidazol~-benzyloxycarbonyl (Z)-L-histidine and N-imidazole-benzyl-L-histidine.
In a particularly preferred aspect of the present invent$on, the replacement amino acids are selectçd from among the group of genetically encoded amino acids, in order to obtain PTH variants that can be produced by application of established, recombinant DNA-based techniques of protein production. In this respect, the replacement amino acids may be selected from among the ~roup consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, a~paragine, glutamine, histidine and proline. In a preferred embodiment of the invention, the replacement amino acids are selected from alanine, valine, leucine, isoleucine, histidine, glutamine, asparagine and proline.
According to specific embodiments of the present invention, X~ and Y~ are preferably histidine and Z~ is preferably leucine.
The amino acid replacements ~erein identified may be introduced into various forms of PTH, i.e. into different ;
.
093/20203 ~ PCT/CA93/00136 g PTH "backqrounds", that in their native state contain the --Arg25Lys26~ysn sequence. The replacement amino acids may be introduced for example into mature forms of vertebrate PTH, including chicken PTH, as well as mammalian PTH
S forms including porcine PTH, rat PTH, bovine PTH and also human PTH. The term "human PTH" refers to the mature form of the hormone, which consists of 84 amino acids arranged in the sequence reported by Kimura et al, 1983, Biochem.
Biophys. Res. Comm., 114(2):493. The terms "human PTH~
and ~hPTH~ are u~ed interchangeably herein. The terms "bovine PTH", "rat PTH" and ~porcine PTH" refer also to ;~
the mature form of the hormone, each of which consist~ of 84 amino acids arranged in the sequence~ reported by ~-Keutmann et al in Current Research on Calcium Regulating Hormones, Cooper, C.W.(Ed.), 1987, University o~ Texas Press, Austin, pp.57-63.
The replace~ent amino acids may also be incorporated into biologically active fragments of mature PTH that -~ -contain the Arg25Lys2qLys27 sequence. "~iologically active fragments~ of PTH comprise at least the first 27 N-terminal residues of a mature PTH species, and most : -~
usually consist of amino acid residues 1-34. Thus, for example, the amino acid replacements herein described can be introduced for example into human PTH(1-34-) and bovine 25 PTH(1-34~, as well as C-terminally extended fragments -~
such as PTHtl-37) and PTH(1-38).
The amino acid replacements herein described can also be incorporated to improve the tryptic stability of PTH analogues and fragments thereof. The term "PTH
analogue" is used herein with reference to Arg25Lys26Lys~-containing forms of PTH haYing an altered amino acid sequence, such as an amino acid substitution at a site other than the Arg25Lys2~ys~ region. Such PTH analogues and fragments thereof include those having substitutions 3S for example at on or both of position~ 8 and 18 whereby -`~
resident methionines are replaced by a hydrophobic amino acid such as norleucine or leucine (see copending W O 93~20203 ~J ~ J ~ ~ PC~r/CA93/00136 , W092/11286 published 9 July 1992 and see Rosenblatt et al, J. Biol. Chem., 1976, 251(1):159); analogues having a substitution at position 12 whereby the resident glycine is replaced by alanine, D-alanine, isobutyric acid, proline, tryptophan or asparagine (see Wingender et al, W090/10067, and Rosenblatt et al U.S. 4,968,669);
analogues having a substitution at position 23 whereby the resident tryptophan is replaced by leucine, N-methyl- -phenylalanine or D-tryptophan (~e Merck & Co., EP
293,159); analogues having a cub~titution at position 32 whereby the resident histidine is replaced by arginine, leucine, lysine or serine (see Wing~nder et al, supra);
and analogues having a substitution at po~ition 34 whereby the resident phenylalanine is replaced by tyrosine.
IS
The replacement amino acids can al~o be incorporated into N-terminally truncated version~ o~ mature PTH and frag~ent~, and analogues thereof, which contain the Arg~LysULysn region. These form~ of PTH have been de~cribed as antagonists of PTH action, and typically lack from 3 to 7 N-terminal residues (see US 4,423,037).
The term "PTH compoundN as used herein thus embraces ArgULys~Lysn-containing forms of PTH, including native P$H forms, N- and C-terminally truncated forms thereof, and analogues of these native and truncated forms.
In a preferred embodiment of the present invention, the replacement amino acids herein described are incorporated into human PTH, to yield hu~an PTH variants that in addition to exhibiting improved stability in the presence of trypsin also exhibit activity comparable to native PTH. According to specific embodiment~ of the invention, such human PTH variants include:
(a) those in which the Arg~ residue alone is replaced by a genetically encoded amino acid selected 35 ~ro~ hi~tidine, tyro~ine, tryptophan, glutamine, -a~paragine, alanine, phenylalanine, leucine and i~oleucine. Specific compounds of the present invention . ~ , W093/20203 PCT/CA93/00136 ; ;
1 1 .. ' include [His~5]hPTH, ~Gln25]hPTH, [Asn~]hPTH, [Phe25~hPTH, ~Trp~]hPTH, [Tyr25]hpTH~ tAla25]hPTH, [Val~]hPTH, ~ -tIle~]hPTH and [Leu~]hPTH, [Thr25]hPTH as well as ~ ~
analogues of these variants in which, for example, the ~;
methionines resident at one or both of positions 8 and 18 are replaced by an amino acid having a hydrophobic side chain, such as leucine; ;-(b) those in which the Lys26 and Lysn residues are each replaced by a genetically encoded amino acid selected from histidine, tryptophan, glutamine, asparagine, leucine and isoleucine Specific compounds of the present invention include [His2~His~]hPTH, ~;
tHis26Leu27]hPTH, ~His~Asn27]hPTH, [His26Gln27~hPTH, ,~
tHis2~Trp~]hPTH, tHi ~Ile271hPTH, tGln2~Gln~]hPTH, tA~n~Asn~]hPTH and ~Gln2~i~n]hPTH, as well as analogues o~ the~e variants in which, for example, the methionines r-~ident at one or both of po~itions 8 and 18 are r-placed by an amino acid having a hydrophobic side chain, ~uch as leucine; ~ `;
(c) those in which the Arg~ residue and the Lys residue are replaced by a genetically encoded amino acid selected from histidine, tryptophan, glutamine, asparagine, leucine and isoleucine Specific compounds -~
of the present invention include tHiS25His26~hPTH, [His~Leu~]hPTH, [His~Asn26~hPTH, [His~Gln26]hPTH, tHis~Trp~hPTH, [His~Ile26~hPTH, tGln~Gln~]hPTH, tAsn~Asn~]hPTH and [Gln~His~hPTH, as well as analogues ;
o~ these variants in which, for example, the methionines ;
resident at one or both of positions 8 and 18 are 3D replaced by an amino acid having a hydrophobic side chain, such as leucine; and -~
(d) those in which the Arg~ residue and the Lys~
residue ~re replaced by a genetically encoded amino acid ~elected from histidine, tryptophan, glutamine, 35 asparagine, leucine and isoleucine Specific compounds -~
of the present invention include tHis~His~]hPTH, tHi~Leu~hPTH, tHis~Asnn~hPTH, [His~Gln~]hPTH, ~
......
,, :',..'.,..'".' W093/20203 J~ ,l PCT/CA93/00136 [His~Trp~]hPTH, [His~Ile~]hPTH, [Gln~Gln~]hPTH, ~Asn~Asn~]hPTH and [Gln~His~hPTH, as well as analogues of these variants in which, for example, the methionines resident at one or both of positions 8 and 18 are S replaced by an amino acid having a hydrophobic side chain, such as leucine.
In a particularly preferred embodiment of the present invention, the PTH variants are variants of hu~an PTH in which each of Ar ~, Lys2~ and Lysn is replaced by a g-netically encoded amino acid selected from histidine, tryptophan, tyrosine, gluta~ine, asparaqine, alanine, valine, leucine and isoleucine. It has been found that such hu~an PTH variants are virtually resistant to trypsin diqestion. It will be appreciated as well that lS such variants will al~o exhibit resistant to attack by enzy~es other than tryp~in which recoqnize basic r~sidues, such as kallikrein and thrombin, and th~ kex enzymes which recognizes dibasic residue~. Speci~ic e~bodiments o~ the present invention include tHis25His2~Leu271hPTH, tHis2sHis~His27~hPTH, tHis2sLeu2qLeu~]hPTH, tHis?sGln~His~]hPTH, ~ ~-;
tHis25Asn26Hisn]hPTH, tHis2STrp26Hisn]hPTH~
tHis~Gln~Leun~hPTH, ~His~Leu~His~]hPTH, [His~Ile~His~hPTH, [Gln~His~His~]hPTH and tGln~Gln2~is~hPTH, as well as analogues of these variants in which, for example, one or both methionines resident at positions 8 and 18 are replaced by an amino acid having a hydrophobic side chain, such as leucine.
As protein products, the PTH variants of the present invention are a~enable to production by the technique of solution- or solid-phase peptide synthesis. The solid phase peptide synthesis technique, in particular, has ~;
~een successfully applied in the production of hu~an PTH
and can ~e used for the production of the PTH var~ants of 3S the pr~s~nt invention (for guidance, see Ximura et al, supra, and sce Pa~rwell et al, Biochem., 1983, 22:2691).
Succes~ with producing human PTH on a relatively large -~
' '"'' ''''.
.' ':''','.
..,,; ~',, scale has been reported by Goud et al in J. Bone Min.
Res., 1991, 6(8):781, incorporated herein by ref~rence. ~ -The synthetic peptide synthesis approach generally entails the use of automated synthesizers and appropriate S resin as solid phase, to which is attached the C-terminal amino acid of the desired PTH variant. Extension of the peptide in the N-terminal direction is then achieved by successively coupling a suitably protected form of the next desired amino acid, using either FMOC- or BOC-based ~0 chemical protocol- typically, until ~ynthQsis i~
complet . Protecting groups are then cleaved from the peptide, usually simultaneously with cleavage of peptide from the re~in, and tbe peptide is then isolated and pur$fied using conventional techniques, ~uch ~s by IS rever~ed phase HPLC using acetonitrile a~ solvent and tri-~luoroacetic acid a~ ion-p~iring agent. Such ~ -procedur-s are generally described in num~rous publication~ and reference ~ay be ~ade, for example, to Stewart and Young, Solid Phase Pept~d~ç ~ynthesis, 2nd 20 Edition, 1984, Pierce Chemical Company, Rockford, ~ i' Illinois. It will be appreciated that the peptide ~;
synthesis approach is required for production of PTH
-v~riant~ which incorporate amino acids that are not genetically encoded.
In a preferred embodiment, the PTH variants of the present invention consist essentially of genetically encoded amino acids, and are produced in accordance with generally established recombinant DNA-based techniques of protein production. More particularly, and in accordance with one aspect of the present invention, such PTH
variants are produced by culturing a cellular host in which DNA coding for the desired PTH variant is stably ~ ;
and expressibly incorporated. Incorporation of the ~ -desired DNA, in expressible form, can be achieved using A'~
35 established procedures, wherein DNA coding for the PTH -~
variant is linked operably with DNA enabling expression o~ the PTH variant-encodin~ DNA, to form a recombinant 4~
`'' ~' W O 93/20203 ~ ~ 14 PC~r/CA93/00136 DNA expression construct which is then introduced into the selected cellular host by DNA-mediated transformation, electroporation or the like. A cellular host having DNA coding for a PTH variant incorporated S "expressibly" therein is characterized by the ability to yield the desired expression product, when cultured appropriately. A cellular host having DNA coding for a PTH variant incorporated Ustably'' is able to retain such DNA during culturing, and to transmit such DNA to its progeny through at lea~t s~veral gQnerations. For eucaryotic cellular hosts, such stability i8 typically conferred by geno~ic integration of the PTH variant- ~ -encoding DNA. In ba~teria, which typically harbour transforminq DNA in the form of autonomously replicating lS pla~m?ds, such ~tability is usually ensured by culturing - -a strain carrying plasmid-conferre~ antibotic resistance in the presence of the antibiotic. ~ -For expression in the cellular host, DNA coding for a selected PTH variant ~ay be obtained using techniques that are well established in the art. For example, a DNA
sequence coding for a given PTH variant may be ~ -s synthesized de novo in accordance with methods standard ~ -in the gene synthesis art. Briefly, this entails the successive 3' to 5' coupling of suitably protected nucleotide reagents in an automated DNA synthesizer, and then the rscovery by gel purification of the deprotected polynucleotide. The block ligation approach may be employed, whereby "blocks" of oligonucleotide pairs, up ~-~
to about 80 nucleotides in length, are prepared and ligated in correct succession by overhang complementarity to assemble the variant-encoding DNA, as described~for example by Wosnick et al in Gene, 1989, 76:153. In an alternative approach, the desired DNA may be synthesized in toto, and then amplified by poly~erase chain reaction 35 (PCR), using the approach described by Barnett et al in t`'~
Nucl. Acids Res., 1990, 18~10):3094.
i y ~. .
It will be appreciated that alternative strategies ~
may also be applied to generate DNA coding for the -desired PTH variant. For instance, DNA coding for human PTH may be obtained and then used as a template e.g.
5 mutagenized site-specifically, to introduce the deslsed -amino acid change at the genetic level. DNA coding for human PTH may be obtained from an appropriate human cDNA
library, from a commercial source or by de novo synthesis according to t~e procedureR outlined above, and in accordance with the PTH-encoding nucleotide sequence reported for example by Hendy et al, Proc. Natl. Acad. ;
Sci. USA, 1981, 78:7365, incorporated herein by - reference, or a PTH-encoding ~quivalent thereof. The PTH-encoding DNA template may be converted to DNA coding ;~
for a PTH variant using the well established oligonucleotide-directed mutagenesis technique, as generally described for example by Xunkel et al, 1985, Proc. Natl. Acad. Sci. USA, 82:488. This technique is conv~niently accomplished with high efficiency using the E. coli-baRed system for synthesis and propogation of the altered gene in an appropriate vector, such as M13mpl8.
Kits useful Eor performing such procedures in vitro are available commercially. Also suitable for obtaining PTH ~ -`
variant-encoding DNA from a PTH-encoding template is the ~-2S related technigue in which site-directed mutagenesis is achieved using a PCR-based approach. One variant of this method, termed "recombinant PCR" is described by Higuchi et al, Nucl. Acids. Res., lg88, 16:7351, and a modified "megaprimer" PCR approach is described in Biotechniques, 1990, 8(1):404.
Once obtained, DNA coding for the desired PTH
variant is incorporated stably and expressibly into a ~-cellular host selected to serve in production of the PTH ~;-variant. A variety of organisms are suitable as hosts -for production of the PTH variants. ThesQ include eukaryotic hosts including yeasts such as Saccharomyces, Pichia and Kluveromyces, filamentous fungus hosts W093/20203 ~ PCT/CA93/00136 including AspergillUs species such as nidulans, niger (or awamori) and oryzae, insect cell hosts, and mammalian cell hosts including the CHo and COS cell lines. The PTH
variants are not dependent on glycosylation for activity, ;~
5 and thus can suitably be produced in bacterial hosts ~ ;
including Streptomyces, Bacillus and, preferably, in E.
coli. Recombinant DNA expression systems and culturing media/protocol~ enabling production in these hosts of a desired protein have already been established, and these 10 syste~s may be employed in the conventional ~anner for the specific purpose of producing PTH variants. E. coli production of PTH variants may be achieved, for examplQ, qv using expression syste~s ba~ed on the lac promoter (~ee Rabbani et al, Biochem., l990, 29:10080) and 15 expression/secretion systems based on the tac promoter (see Wong et al, EP 357,391). Yeast expre~sion may be achieved using expression systems based for example on the expre~sion controlling regions of the alpha-l mating ~ ~ ~
f~ctor gene a~ described by Gautvik et al in W088/03165.~ ~`
20 Production in Aspergillus may be achieved using secretion-~ ~
syste~s based on exprescion controlling region~ of the A. ~--nidulans alcA gene or the A. niger glucoa~ylase gene, as described for example by Gwynne et al in W086/06097. ~ ;
The P$H variant generated upon culturing of the ;~
production strain is extracted and purified using techniques that are al50 established in the art. In t general, the human PTH variants have characteristics that are similar generically to those exhibited by hu~an PTH, and may therefore be extracted and purified in 30 substantially the sa~e manner. Like PTH, the variants have a net positive charge at neutral pH (pI o~ about 9.3) and can be purified therefore by ion exchange chromatography, e.g. using cation exchange columns. The PTH variants are also, like PTH, hydrophobic in nature, 35 and may therefore be purified by hydrophobic interaction chro~tography e.g. on columns having a phenyl-Sepharose ~atrix. Al~o, of course, molecular sieves ~ay be u~ed to W093/20203 ' ~ PCT/CA93/00136 separate PTH variants from ot~er proteins unrelated by .
size, and affinity columns may be employed which comprise ;
PTH affinity agents such as hydroxyapatite or PTH
antibody. Preferably, purification of the PTH variant is S achieved by applying the protein mixture to a cation exchange column e.g. S-Sepharose, and then applying the eluted retentate to a column having a hydrophobic matrix e.g. a column havinq a phenyl, octyl or butyl side chain Quch a~ phenyl-Sepharose, phenyl-Superose, octyl-10 Sep~aro~e or butyl 650M. The retentate eluted ~rom the ;-hydrophobic matrix can then be subjected to final purification using reversed phase high performance liguid ~
chromatography (HPLC). ;
While the tryptic instability of human PTH typically lS demand~ that great care be taken during purification to guard against contamination by tryp~in in glassware and during handling, extraction and purification of the PTH
variants of the invention requires less stringent control mea~ure~. It is nevertheless desirable to exercise such control, in keeping with good laboratory and manufacturing practise.
For therapeutic use, a PTH variant is desirably purified to the extent that it migrates as a single peak on reversed phase HPLC, and exhibits a single band on polyacrylamide gel electrophoresis in the presence of SDS. Once purified, the PTH variant may be formulated to provide pharmaceutical composition~ ~uitable for treating the various clinical conditions for which PTH therapy is indicated. Compositions containing PTH variant are admini-~tered desirably to treat bone disordexs such as o~teoporosi~ and other osteopenic conditions, and for these purpo~es are suitably formulated either as injectables or ingestibles or for nasal insufflation, in accordance with established practi~e~ of protein drug formulation. Sterile injectable composition~ are particularly useful, and will generally compri~e an effective dose of the PTH variant, in admixutre with W093/20203 ~ '~ ~4 ~ PCT/CA93/00136 normal saline and suitable solubilizing agent e.g. dilute acetic acid. The PTH variant may alternatively be applied topically, as a cream, lotion, ointment or as an aerosol, to treat psoriasis and related skin disorders.
A suitable cream comprises an effective dose of the PTH
variant, in combination with carriers of standard composition e.g. in a triglyceride base.
A therapeutically effective dose of PTH variant, i.e. a do~e of PTH variant effective to treat a given clinical condition will depend of course on the nature and severity of the condition, and on such other factors as are normally considered and evaluated in clinical trials and by the attending physician. For treating osteoporosis, the PTH variant iB administred in amounts large enouqh to stimulate bone remodelling, but not so large as to cause net bone re~orption or sustained increase in serum calcium levels. Reference may be made to US patent 4,698,328 for guidance on the admini~tration of PTH to treat osteoporosis. Using the effective PTH
doses in a given clinical situation for guidance, the dose of PTH variant required to elicit a similar effect can be calculated based on the relative activity of the P~H variant. For example, ~His~]hPTH, [His~His~$eu~hPTH
and hPTH are substantially equipotent, and effective 25 doses of these PTH variants are thus similar to those of ;~
hPTH. It iB expected that the improved stability in the presence of trypsin of the PTH variants will provide for extended in vivo half-life, and thus somewhat smaller doses may be used or similar do es may be administered less frequently. It is anticipated that dosage sizes in the range from 0.05 ~g/kg to about l,000 ~g/kg, for exa~ple in the range from O.l ~g/kg to lO0 ~g/kg, and more suitably about 1-10 ~g/3cg will be clinically useful.
Like PTH, the PTH variants may be administered in 35 combination with other agents useful in treating a given --~
clinical condition. When treating osteoporosis and other -bone-related disorders for example, the PTH ~ariants may :
W093/20203 t,~ PCT/CA93/00136 be administered in conjunction with a dietary calcium ~ ~-supplement or with a vitamin D analogue (see US
4,698,328). Alternatively, the PTH variant may be administered~ preferably using a cyclic therapeutic ~;;
regimen, in combination with bisphosphonate~,as described - .... ,;
for example in US 4,761,406, or in combination with one or more bone therapeutic agent~ such as calcitonin and estrogen.
Exam~les ~-The example~ which follow describe production of huaan PTH and human PTH variants. Production of these proteins was achieved using, as a matter of convenience only, an E. coli-basQd system ~ubstantially as described by Wong and Sutherland in European patent application 89308?53.6 (publi~hed as EP357,391 on 7 March l990), the contents of which are incorporated herein by reference.
Thi~ sy~tem makes use of the commonly available E. coli JH101 strain as host and employs as vector a pUCl8 deriv~tive, designated pX. As is shown in Figure l, pX
incorporates the par element of pSClOl to enhance freguency of plasmid transmission, the lacIq gene of pMMB22 to enable overproduction of the lac repressor, and a PTH-excretion cassette. Incorporated in the excretion cassette is human PTH-encoding DNA that was synthesized using the block ligation technique reported by Wosnick et al, supr~, and in accordance with the PTH-encoding nucleotide sequance reported by Hendy et al, supr~.
Fused 5' of, and precisely to, the PTH-encoding DNA is the ~ignal ~equ~nce of the E. coli ompA gene, which is `?
capable of directing the PTH portion of the expression product across the host inner membrane, which migrates ultimately to the culturing medium. For regulated expression of the coding region, the plasmid operably -~
incorporates the tac promoter, the lac operator and a 3S con en~us ribo~omal binding site. Transcriptional ter~in~tion is controlled by the E. coli trpA gene terminator, and translational stop codons are provided in ;~
W093/~0203 ;~ 20 PCT/CA93/00136 all three reading frames, immediately 3' of the PTH~
encoding DNA.
Thus, the pX expression vector, used for the production of human PTH and PTH variants, is -5 substantially the same as that described by Wong and -~
Sutherland, supra, except that the multiple cloning site downstream of the PTH gene contains cleavage sites for - -~
the restriction enzymes ClaI, BamHI, XbaI, StuI and PstI, in the order indicated on Figure 1. The precise nucleotide seguence o~ the PTH-encoding region of the excretion cassette is illustrated in Figure 2.
Exam~le 1 - Production of human PTM(1-84) . ~
Plasmid pX was transfor~ed into competent E. coli JMlOi using standard procedures. Positive transformants were indentified following growth overnight at 30C on plates containing 2YT/agar and 70~g/ml ampicillin. PTH-producing transformants were then examined ~or PTH
activity, following growth in shake flasks, by IRMA
analysis of conditioned medium. and frozen stocks of the sQlected transformants were subsequently prepared by mixing an equal volume of the shake flask culture with sterile glycerol to yield 50%(v/v) glycerol stocks.
These stocks were subsequently stored at -80C. When needed, transformants were recovered from the frozen -stock by scraping, and were then streaked on ampicillin-containing plates of 2YT/agar.
To produce human PTH, freshly plated transformants were picked as single colonies and then inoculated into 50ml Erlenmeyer flasks containing 15ml of a liquid medium which contained 2YT, glucose and ampicillin in the standard mixture. Following overnight growth with ~-shaking at 30C, the cultures were diluted 20-fold with fresh medium, and then grown for three hours at 30-C with shaking. Expression of the PTH-encoding DNA was then de-repressed by addition of l.OmM IPTG. After growth for , ' ,''"
'- .,., ' - -.
... .
w093/20203 PCT/CA93/00136 four hours in the presence of IPTG, the culture was -cooled to 4C and centrifuged. The supernatant was then harvested and human PTH contained therein was recovered -and assayed for PTH activity. .4 S To obtain sufficient quantities of human PTH(1-84) and the PTH variants for purification and bioassay, larger volumes of conditioned ~edia werQ collect~d. In particular, freshly plated transfor~ants were picked as single colonies and then inoculated into 500ml flasks containing 200~1 of the ~edium described above.
Following overnight growth with shaking at 30-C, the cultures were inoculated into 2L bioreactor~ containing l.SL of the liquid medium, and then grown for S hours ~t 30-C with stirring. Expression of the PTH- or PTH
1S variant-encoding DNA was then induced by add~tion of 1.O~M IPTG. After growth for 3-4 hours in the presence of IPTG, the culture was cooled to 4-C and centrifuged.
The supernatant was then harvested, and the PTH or PTH
variant contained therein was purified in the manner described in Example 5.
The examples which follow describe production of PTH
variants. To obtain DNA coding for these variants, the in vitro site-directed mutagenesis technique described by Kunkel et al, supra, was applied. To perform this procedure there was first obtained plasmid RX which is an Ml3mpl8-based version of the excretion cassette on pX, carrying the PTH-encoding DNA as a promoterless NruI/XbaI
insert. Plasmid RX thus served as the template for conducting mutagenesis on the PTH-encoding DNA, in order to gQnerat~ DNA coding for a desired PTH variant. The particular mutagenesis strategy is described in the ~ -examples below.
Exa~Dle 2 - Production of a tHis~] variant of PTH
To provide DNA coding for a PTH variant in which Arg~ is replaced by histidine, plasmid RX was first W093/20203 '~J~ y 22 PCT/CA93/00136 recovered in single stranded form and about l~g thereof was incubated, at 85OC in Hin buffer, with about lOOng of a mutagenic oligonucleotide capable of hybridizing specifically to that region of the PTH gene containing -the Arg~ codon. The specific sequence of the oligonucleotide, designated Pl, is shown below where underlining indicates the codon change relative to the PTH-encoding template shown in Figure 2:
0 Pl oligos 5' CAGCTSCrSi~iC~GCCASSCrAC 3' t-~pl~t~: 3'....... CTOCAACAASCOCTOCCTAACATC........ 5~ ~ :
` ' After slow cooling, the annealed fragment was tre~ted with DNA polymer~e I (Xlenow) in the presence lS of all four dNTPs, for about 2 hour~ at 37C and then for 4 ho~rs at roo~ temperature, in order to form the full length double-stranded plas~id, d~signated pRXPl.
Co~pet~nt ho~t JMlOl was then tran~formed by pRXPl, and plaques were screened by restriction digest analysis and ~ ;
20 by DNA sequencing to select those carrying the desired ~
mutation. ~ ;
pRXPl is then digested with NruI and XbaI and the re~ulting small fragment is isolated by low melting point ~;
agarose. Plasmid pX is similarly digested, and the large 2S NruI/XbaI fragment is isolated. The relevant isolated fragments are then ligated, to form plasmid pXPl, which ;-~
carries DNA coding for [His~]hPTH. This was confirmed by ;
restriction digest analysis and DNA sequencing.
Competent E. coli JMlOl was transformed with pXPl - `~
and the transformants were then ~elected in accordance with the procedures outlined in Example l. Supernatant ~ ' containing the [His~PTH was then obtained for subsequent -...
purification by culturing the pXPl transformant, in the ~ -manner described by example l. ~--~
. . . ~ . .-. ~
.... .
.~':' :,',..
'~' '.' "'"' .: , , , - ,.~' -: . :
'J
Exam~le 3 - Production of a ~His2sHis26Leu~] variant of PTH
In a manner similar to that described in Example 2, there was obtained DNA coding for a human PTH variant in which the native sequence Arg25Lys2~Lysn is replaced by the sequence His~His26Leu~. In particular, single stranded pRX was incubated with an oligonucleotide having the sequence provided below, where underlining indicates the ., codon change relative to the native PTH-encoding template -shown in Figure 2:
P2 oligo 5 ' CATCCTGCACCACGTGCTGCACCCASTC~AC~C~ 3 ~ :
PTH t~pl~t- 3 ' . . .CSACCACC~CCAACA~TCCCTCCCTAACATCACA. . . 5 ~ ~ .
:
A double stranded plasmid carrying the desirQd codon replacements, designated p~XP2, is then cut with NruI/XbaI and the isolated small fragment is ligated with the large fragment of NruI/XbaI-digested pX. E. coli was then transformed by the resulting plasmid pXP2, and the transformant wa~ cultured in the manner outlined in Example l to yield supernatant containing -tHis~His~Leu~]hpTH.
Exam~le 4 - Production of additional PTH variants In the manner substantially as described above in example 3, supernatants containing additional PTH
variants are obtained by culturing E. coli transformants habouring PTH variant-encoding DNA. To generate the variant-encoding DNA, DNA coding for native hPTH (Example
Field of ~he Invention ~-This invention relates to variants of parathyroid hormone, to the produetion of such variant~ particularly S via reeombinant DNA teehnology and to the use of such ~
variants as therapeutie agents, for example in the ~ :
treatment of oQteoporosis.
Baekaround to the Invention Parathyroid hormone (PTH) is a seereted, protein produet of ma~malian parathyroid glands that regulates ealeium ho~eostasis through its aetion on various tissues, ineluding bone and vaseular tissue. Reaeareh into the physiologieal role o~ PTH has identified elinieally relevant effeets on bone metabolism, there being. so~e elinieal evidenee that PTH may be useful in the treat~ent of ost~oporo~is and related o~teopenie a~flietions. An effeet on vaseular tissue and on keratinoeyte growth has also been noted.
To obtain PTH in the a~ounts required for elinieal investigations and for eommereial purposQs, reeombinant DNA-based teehnique~ have been sueeessfully applied in -~
the produetion of sueh mammalian PTH speeies as human ;~
PTH, bovine PTH, poreine PTH and rat PTH, whieh in their mature form all eontain 84 amino aeids arranged in a speeies-speeifie sequenee. An understanding of the strueture of these proteins, and partieularly human PTH --and bovine PTH, has lead al~o to the diseovery that PTH
aetivity ean be attributed to the first 34 N-terminal residues of the mature hormone. This ha~ allowed for the production of biologically active PTH fragments using the ~olid phase technique of peptide synthe~is, to meet PTH ~-demand.
In the interest of furthering development of PTH as a pharmaceu~ical product, it would be desirable to provide stability-enhanced forms of PTH that are better ~uited for hu~n administration. It is known from studies with serum-derived PTH samples, and from ' " "..
,, " ..
:
~ ~ j~J !~
experience with recombinant PTH production, that the ;
hormone is particularly vulnerable to protease digestion. ~:
PTH production in yeast, for example, has shown that PTH
is degraded by the yeast-produced kex enzyme, which -recognizes dibasic residues. When the lysine residue at po~ition 26 of PTH is replaced with glutamine, however, -~
proteolytic degradation of PTH by the yeast is reportedly reduced (~ee Reppe et al, J. Biol. Chem., 1991, 266:14198). Other studie~ targetting thi~ general region o~ the PTH molecule have indicated that amino acid replacement can cause significant decline in PTH ;~
activity. Conversion of the ly~ine at position 27 for exa~pl~, resulted in a marked decline in PTH activity (see Wingender et al, WO 90/10067) as did derivatization lS of the arginine residue~ at positions 25 and 20 (see Rosenblatt et al, 1978, Biochemistry, 17:3188).
It is an object of the present invention to provide a novel variant of parathyroid hormone.
It is another object of the present invention to 20 provide a parathyroid hormone variant that exhibits -improved stability in the presence of proteolytic enzy~es, e~pecially trypsin and trypsin-like enzymes.
It is another object of the present invention to ~-provide a parathyroid hormone variant that exhibits both 25 improved stability in the presen e of proteolytic enzymes -~
and a bioactivity that is comparable to parathyroid hormone.
It is another object of the preaent invention to `~
prov~de a pharmaceutical composition comprisinq a parathyroid hormone variant exhibiting improved stability and comparable activity, for use in therapeutic applications.
It is another object of the present invention to provid~ a process for producing a stability-enhanced -variant of parathyroid hormone.
5~= '~ ' ' : ` ~
WOg3/20203 PCT/CA93/00136 ~, ,' 3 '~' The present invention provides variants of ~-' parathyroid hormone that are altered structurally to confer enhanced stability in the presence of proteolytic '~-enzymes such as trypsin and the so-called trypsin-like S enzymes that cleave at internal arginine and lysine re~idues. Although numerous putative tryp~in cleavage ', sites reside in native parathyroid hormone, it has surprisingly been found that substantial resistance to tryptic digestion is conferred when the hormone is 10 altered within a single region con~titut-d by re~idues ~-, Arg~Lys~Ly~n. It has further been found that variants -having activity comparable to native parathyroid hormone ''~
are generated whQn replacemQnt amino acids are se,lected appropriately. Thus, the PTH variants of the present lS invention are well suited for therapeutic applications ~-~
and are b~tter adapted to survive expo~ure particularly to tryp~in and trypsin-like enzy~es while in the p~ripheral circulation and al~o during their production, , handling and storage. ;~
~ccording to one aspect of the pre~ent invention, there is provided a stability-enhanced variant of a parathyroid hor~one co~pound harbouring the region -~
Arg25Lys~Lysn, in which said region is replaced in said variant by an amino acid sequence selected fr~m~
(a) X~Lys26Lys~; -(b) Arg25y26z~ "
(c~ X2SY~Lys~; , (d) X~Lys~Z~; and '`"~
(e) XUY~Z~
wherein X, Y and Z are independently selectad trypsin-insensitive amino acids i.e. amino acids other than lysine and arginine. - '-In a preferred embodimen~ of the invention, the ' ~, replacement a~ino acids X, Y and Z are selected to yield ' ','-3S variant~ o~ hu~n PTH that in addition to exhibiting enh~nced stability in the presence of tryp~in, al~o '~,~
exhibit an activity comparable to native human PTH. In '~' ;, :' .
W093/20203 ~ ~S~t~ Y PCT/CA93/00136 :s specific embodiments of the invention, the replacement amino acids are selected from among the genetically encoded amino acids, to permit production of the variants -via recombinant DNA-based techniques.
According to another aspect of the pre~ent invention, there is provided a pharmaceutical composition, comprising a therapeutically effective amount of a parathyroid hormone variant of the present invention and a pharmaceutically acceptable carrier.
According to another aspect of the present invention, there is provided a method for treating a ma~oal, which comprises the step of administering to a ~a~oal in need thereof a pharmaceutical composition lS co~pri~ing a therap~utically effective amount of a parathyroid hormone variant of the present invention and a phar~aceutically acceptable carrier.
According to another aspect of the present invention, there is provided a process for producing a ~ ~-parathyroid hormone variant that exhibits enhanced ~tability in ~he presence of trypsin, which comp~ises the step of culturing a cellular host having incorporated expressibly therein a DNA molecule which codes for a PTH - ~-variant consisting only of genetically encoded amino 25 acids. -The invention and its preferred embodiments are now described in greater detail with reference to the accompanying drawings, in which:
Brief Reference to the Drawinas Figure l illustrates a recombinant DNA plas~id harbouring human PTH-encoding DNA, for expression in an E. coli host; and Figure 2 illustrates the sequence of the human PTH-encoding DNA incorporated on the plas~id of Figure 1, and 3S provides the sequence of human PTH with the Arg~Lys~Lys~
region identified by boxing.
Detailed Descri~tiOn of the Invention and Prefer~d Embodiments The invention relates to parathyroid hormone ;~
variants that exhibit improved stability in the presence ~
5 of trypsin and, in accordance with a preferred aspect of ~ -the invention, also exhibit activity at least similar to native PTH. In the pre~ent ~pecification, PTH activity is defined in the context o~ the o~teo~arcoma-based adenylate cycla~e a~ay employed conventionally in the art. Briefly, this a~say provide~ an in vitro determination o~ tbe extent to hich PTH stimulates adenylate cyclase activity in rat osteosarcoma cells of the `UMR' lineage, and thus provides an indication of PTH
effects on bone ti~ue in vi~o. Protocols for conducting the a~ay have been de5cribed by Rodan et al, 1983, J. -Clin. Inve~t., 72:15~1 (in which the osteo~aracoma cells of the ROS lineage are employed) and by Rabb~ni at al, - --1988, Endocrinol., 123:2?09 (which employ~ the line UMR~
106). PTH variants that exhibi~, in the UMR-ba~ed assay, an EC~ of at least 1,000 nM i.e. 1,000 nM or lower, are --herein characterized as having activity Usimilar~ to native PTH; a variant having an EC~ of lOOnM or lower is characterized as having activity Usubstantially similar"
to native PTH; and a variant having an EC~ of 20nM and lower is characterized as having an activity that is "comparable" to native PTH. The term EC~ refers to the concentration of PTH or variant effective for half-maximal stimulation of adenylate cyclase activity in the UMR-ba~ed as~ay.
PTH variants having an "improved stability in the presence of trypsinH are degraded ~y trypsin a~ a rate that is slower than a similarly treated native PTH
control. An assay suitable for identifying a reduced tryptic digestion rate entails a two step procedure, in 3S which a PTH variant and a native P~H control are ~-~eparatQly incubated witb trypsin for a de~ined period, ~-and are then assayed for activity in tbe osteosarcoma~
~':''.'~ ....
W093/20203 ~ j PCT/CA93/00136 based assay just described Protocols suitable for assaying trypsin sensitivity are described in the literature and are outlined in the examples herein In ~;~
this assay, variants of human PTH, for example, will exhibit an activity that is at least greater than a similarly treated native human PTH control In a quantitative context, the PTH variants ~ay be characterized ac having an improved stability in the -pr-sence of trypsin if the variant exhibits an EC~ as determined in the UMR-based adenylate cyclase a~say that is at least lower than a similarly treated native PTH
counterpast Under conditions of the assay exemplified her~in, for instance, native human PTH xhibits an EC~ of about 20nM or greater following trypsin incubation ~Stab~lity improved" human PTH variants are accordingly characterized by an EC~ of lower than 20nM, when assayed under these particular conditions In the present specification, a~ino acids are a~igned numerals to identify their location relative to the N-ter~inal amino acid of mature PTH For consistency and as is conventional in the art, amino acids are assigned the same positional number when present in the context of N-terminally truncated or extended forms of PTH.
In accordance with the present invention, PTH --variant~ exhibiting improved stability in the presence of trypsin are obtained when the Arg~Lys~Lysn region of PTH
i5 replaced with an amino acid sequence selected from (a) X2SLys~Lys27;
(b) Arg25y~zn (c) X25y2~LySn;
(d) X75Lys~Z~; and (e) X2sy26zn wherein X, Y and Z are independently selected, tryp~in-in~en~itive a~ino acids, i e a~ino acids other th~n ~rginine and lysine wo 93/20203 ~ 9 PCT/CA93/OOt36 It has surprisingly been found that measurably improved stability in the presence of trypsin is realized when the Arg25Lys26Lys27 region is altered by replacement merely of the Arg25 residue. Thus, according to one embodiment of the present invention, there is provided a PTH variant having improved stability in the presence of ~;
trypsin, in which Arg25 is replaced by an amino acid other than arginine and ly~ine.
According to another embodimsnt of tbs present invention, PTH variants exhibiting improved stability in the presence of tryp~in are obtained by replacing both ~ -Arg~ and Lys26 with independently selected amino acids ;
other than arginine and lysine. -According to another embodiment of the present -~
IS invsntion, PTH variants exhibiting improved stability in the pressnce of trypsin are obtained by replacing both .
the Arg25 and Lys~ with independently selected amino acids other than arginine and lysine.
According to another embodiment of the present ~ ~
20 invention, PTH variants exhibiting improved stability in i --the pre~ence of trypsin are obtained by replacing both ~-the Lys26 and Lysn with independently selected a~ino acids other than arginine and lysine.
PTH variants in which the entire Arg~Lys26Lys27 region 25 is replaced have been found to exhibit substantial ~ -resistance to tryptic digestion. According to a preferred ~-embodiment of the present invention therefore, the PTH
variants of the present invention are obtained by replacing each of the a~ino acids Arg25Lys26Lysn with an 30 independently selected amino acid other than arginine and ~ ~-lysine.
The replacement amino acids X, Y and Z in the above ~ -formulae are trypsin-insensitive amino acids and may be ~-~
cho~en from among the various ~ynthetic amino acids and ~ -3S th~ naturally occurring amino acids other than L-arginine ~nd L-ly~ine. ~n a preferred a~pect o~ the pre~ent ~
invention, the replace~ent amino acids X, Y and Z are ~ -.- ~
WOg3/20~03 ~ ~ PCT/CA93/00136 selected with a view to generating variants that exhibit activity comparable to native PTH. For this purpose, the ~-replacement amino acids are selected from among those amino acids which preserve the same configuration in this region of the PT~ molecule. More particularly, t~e replacement amino acids are most desirably selected from among those amino acids having either neutral or positively charged amino acid side-chains. UQeful amino acid replacements having neutral side chains include glycine, alanine, valine, leucine, isoleucine, ~erine, threonine, a~paragine, glutamine, phenylalanine, cysteine, tryptophan, tyrosine, methionine, proline, as well as the ~ynthetic analo~ues thereof, such a~
norleucine, norvaline, cyclohexylalanine, etc. Amino acids h~ving positively charged side chaina include hi~tidine and synthetic histidine analogue~, such as D-hi~tidine, l-methyl-L-histidine, 3-methyl-L-hist~dine, N-imidazol~-benzyloxycarbonyl (Z)-L-histidine and N-imidazole-benzyl-L-histidine.
In a particularly preferred aspect of the present invent$on, the replacement amino acids are selectçd from among the group of genetically encoded amino acids, in order to obtain PTH variants that can be produced by application of established, recombinant DNA-based techniques of protein production. In this respect, the replacement amino acids may be selected from among the ~roup consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, a~paragine, glutamine, histidine and proline. In a preferred embodiment of the invention, the replacement amino acids are selected from alanine, valine, leucine, isoleucine, histidine, glutamine, asparagine and proline.
According to specific embodiments of the present invention, X~ and Y~ are preferably histidine and Z~ is preferably leucine.
The amino acid replacements ~erein identified may be introduced into various forms of PTH, i.e. into different ;
.
093/20203 ~ PCT/CA93/00136 g PTH "backqrounds", that in their native state contain the --Arg25Lys26~ysn sequence. The replacement amino acids may be introduced for example into mature forms of vertebrate PTH, including chicken PTH, as well as mammalian PTH
S forms including porcine PTH, rat PTH, bovine PTH and also human PTH. The term "human PTH" refers to the mature form of the hormone, which consists of 84 amino acids arranged in the sequence reported by Kimura et al, 1983, Biochem.
Biophys. Res. Comm., 114(2):493. The terms "human PTH~
and ~hPTH~ are u~ed interchangeably herein. The terms "bovine PTH", "rat PTH" and ~porcine PTH" refer also to ;~
the mature form of the hormone, each of which consist~ of 84 amino acids arranged in the sequence~ reported by ~-Keutmann et al in Current Research on Calcium Regulating Hormones, Cooper, C.W.(Ed.), 1987, University o~ Texas Press, Austin, pp.57-63.
The replace~ent amino acids may also be incorporated into biologically active fragments of mature PTH that -~ -contain the Arg25Lys2qLys27 sequence. "~iologically active fragments~ of PTH comprise at least the first 27 N-terminal residues of a mature PTH species, and most : -~
usually consist of amino acid residues 1-34. Thus, for example, the amino acid replacements herein described can be introduced for example into human PTH(1-34-) and bovine 25 PTH(1-34~, as well as C-terminally extended fragments -~
such as PTHtl-37) and PTH(1-38).
The amino acid replacements herein described can also be incorporated to improve the tryptic stability of PTH analogues and fragments thereof. The term "PTH
analogue" is used herein with reference to Arg25Lys26Lys~-containing forms of PTH haYing an altered amino acid sequence, such as an amino acid substitution at a site other than the Arg25Lys2~ys~ region. Such PTH analogues and fragments thereof include those having substitutions 3S for example at on or both of position~ 8 and 18 whereby -`~
resident methionines are replaced by a hydrophobic amino acid such as norleucine or leucine (see copending W O 93~20203 ~J ~ J ~ ~ PC~r/CA93/00136 , W092/11286 published 9 July 1992 and see Rosenblatt et al, J. Biol. Chem., 1976, 251(1):159); analogues having a substitution at position 12 whereby the resident glycine is replaced by alanine, D-alanine, isobutyric acid, proline, tryptophan or asparagine (see Wingender et al, W090/10067, and Rosenblatt et al U.S. 4,968,669);
analogues having a substitution at position 23 whereby the resident tryptophan is replaced by leucine, N-methyl- -phenylalanine or D-tryptophan (~e Merck & Co., EP
293,159); analogues having a cub~titution at position 32 whereby the resident histidine is replaced by arginine, leucine, lysine or serine (see Wing~nder et al, supra);
and analogues having a substitution at po~ition 34 whereby the resident phenylalanine is replaced by tyrosine.
IS
The replacement amino acids can al~o be incorporated into N-terminally truncated version~ o~ mature PTH and frag~ent~, and analogues thereof, which contain the Arg~LysULysn region. These form~ of PTH have been de~cribed as antagonists of PTH action, and typically lack from 3 to 7 N-terminal residues (see US 4,423,037).
The term "PTH compoundN as used herein thus embraces ArgULys~Lysn-containing forms of PTH, including native P$H forms, N- and C-terminally truncated forms thereof, and analogues of these native and truncated forms.
In a preferred embodiment of the present invention, the replacement amino acids herein described are incorporated into human PTH, to yield hu~an PTH variants that in addition to exhibiting improved stability in the presence of trypsin also exhibit activity comparable to native PTH. According to specific embodiment~ of the invention, such human PTH variants include:
(a) those in which the Arg~ residue alone is replaced by a genetically encoded amino acid selected 35 ~ro~ hi~tidine, tyro~ine, tryptophan, glutamine, -a~paragine, alanine, phenylalanine, leucine and i~oleucine. Specific compounds of the present invention . ~ , W093/20203 PCT/CA93/00136 ; ;
1 1 .. ' include [His~5]hPTH, ~Gln25]hPTH, [Asn~]hPTH, [Phe25~hPTH, ~Trp~]hPTH, [Tyr25]hpTH~ tAla25]hPTH, [Val~]hPTH, ~ -tIle~]hPTH and [Leu~]hPTH, [Thr25]hPTH as well as ~ ~
analogues of these variants in which, for example, the ~;
methionines resident at one or both of positions 8 and 18 are replaced by an amino acid having a hydrophobic side chain, such as leucine; ;-(b) those in which the Lys26 and Lysn residues are each replaced by a genetically encoded amino acid selected from histidine, tryptophan, glutamine, asparagine, leucine and isoleucine Specific compounds of the present invention include [His2~His~]hPTH, ~;
tHis26Leu27]hPTH, ~His~Asn27]hPTH, [His26Gln27~hPTH, ,~
tHis2~Trp~]hPTH, tHi ~Ile271hPTH, tGln2~Gln~]hPTH, tA~n~Asn~]hPTH and ~Gln2~i~n]hPTH, as well as analogues o~ the~e variants in which, for example, the methionines r-~ident at one or both of po~itions 8 and 18 are r-placed by an amino acid having a hydrophobic side chain, ~uch as leucine; ~ `;
(c) those in which the Arg~ residue and the Lys residue are replaced by a genetically encoded amino acid selected from histidine, tryptophan, glutamine, asparagine, leucine and isoleucine Specific compounds -~
of the present invention include tHiS25His26~hPTH, [His~Leu~]hPTH, [His~Asn26~hPTH, [His~Gln26]hPTH, tHis~Trp~hPTH, [His~Ile26~hPTH, tGln~Gln~]hPTH, tAsn~Asn~]hPTH and [Gln~His~hPTH, as well as analogues ;
o~ these variants in which, for example, the methionines ;
resident at one or both of positions 8 and 18 are 3D replaced by an amino acid having a hydrophobic side chain, such as leucine; and -~
(d) those in which the Arg~ residue and the Lys~
residue ~re replaced by a genetically encoded amino acid ~elected from histidine, tryptophan, glutamine, 35 asparagine, leucine and isoleucine Specific compounds -~
of the present invention include tHis~His~]hPTH, tHi~Leu~hPTH, tHis~Asnn~hPTH, [His~Gln~]hPTH, ~
......
,, :',..'.,..'".' W093/20203 J~ ,l PCT/CA93/00136 [His~Trp~]hPTH, [His~Ile~]hPTH, [Gln~Gln~]hPTH, ~Asn~Asn~]hPTH and [Gln~His~hPTH, as well as analogues of these variants in which, for example, the methionines resident at one or both of positions 8 and 18 are S replaced by an amino acid having a hydrophobic side chain, such as leucine.
In a particularly preferred embodiment of the present invention, the PTH variants are variants of hu~an PTH in which each of Ar ~, Lys2~ and Lysn is replaced by a g-netically encoded amino acid selected from histidine, tryptophan, tyrosine, gluta~ine, asparaqine, alanine, valine, leucine and isoleucine. It has been found that such hu~an PTH variants are virtually resistant to trypsin diqestion. It will be appreciated as well that lS such variants will al~o exhibit resistant to attack by enzy~es other than tryp~in which recoqnize basic r~sidues, such as kallikrein and thrombin, and th~ kex enzymes which recognizes dibasic residue~. Speci~ic e~bodiments o~ the present invention include tHis25His2~Leu271hPTH, tHis2sHis~His27~hPTH, tHis2sLeu2qLeu~]hPTH, tHis?sGln~His~]hPTH, ~ ~-;
tHis25Asn26Hisn]hPTH, tHis2STrp26Hisn]hPTH~
tHis~Gln~Leun~hPTH, ~His~Leu~His~]hPTH, [His~Ile~His~hPTH, [Gln~His~His~]hPTH and tGln~Gln2~is~hPTH, as well as analogues of these variants in which, for example, one or both methionines resident at positions 8 and 18 are replaced by an amino acid having a hydrophobic side chain, such as leucine.
As protein products, the PTH variants of the present invention are a~enable to production by the technique of solution- or solid-phase peptide synthesis. The solid phase peptide synthesis technique, in particular, has ~;
~een successfully applied in the production of hu~an PTH
and can ~e used for the production of the PTH var~ants of 3S the pr~s~nt invention (for guidance, see Ximura et al, supra, and sce Pa~rwell et al, Biochem., 1983, 22:2691).
Succes~ with producing human PTH on a relatively large -~
' '"'' ''''.
.' ':''','.
..,,; ~',, scale has been reported by Goud et al in J. Bone Min.
Res., 1991, 6(8):781, incorporated herein by ref~rence. ~ -The synthetic peptide synthesis approach generally entails the use of automated synthesizers and appropriate S resin as solid phase, to which is attached the C-terminal amino acid of the desired PTH variant. Extension of the peptide in the N-terminal direction is then achieved by successively coupling a suitably protected form of the next desired amino acid, using either FMOC- or BOC-based ~0 chemical protocol- typically, until ~ynthQsis i~
complet . Protecting groups are then cleaved from the peptide, usually simultaneously with cleavage of peptide from the re~in, and tbe peptide is then isolated and pur$fied using conventional techniques, ~uch ~s by IS rever~ed phase HPLC using acetonitrile a~ solvent and tri-~luoroacetic acid a~ ion-p~iring agent. Such ~ -procedur-s are generally described in num~rous publication~ and reference ~ay be ~ade, for example, to Stewart and Young, Solid Phase Pept~d~ç ~ynthesis, 2nd 20 Edition, 1984, Pierce Chemical Company, Rockford, ~ i' Illinois. It will be appreciated that the peptide ~;
synthesis approach is required for production of PTH
-v~riant~ which incorporate amino acids that are not genetically encoded.
In a preferred embodiment, the PTH variants of the present invention consist essentially of genetically encoded amino acids, and are produced in accordance with generally established recombinant DNA-based techniques of protein production. More particularly, and in accordance with one aspect of the present invention, such PTH
variants are produced by culturing a cellular host in which DNA coding for the desired PTH variant is stably ~ ;
and expressibly incorporated. Incorporation of the ~ -desired DNA, in expressible form, can be achieved using A'~
35 established procedures, wherein DNA coding for the PTH -~
variant is linked operably with DNA enabling expression o~ the PTH variant-encodin~ DNA, to form a recombinant 4~
`'' ~' W O 93/20203 ~ ~ 14 PC~r/CA93/00136 DNA expression construct which is then introduced into the selected cellular host by DNA-mediated transformation, electroporation or the like. A cellular host having DNA coding for a PTH variant incorporated S "expressibly" therein is characterized by the ability to yield the desired expression product, when cultured appropriately. A cellular host having DNA coding for a PTH variant incorporated Ustably'' is able to retain such DNA during culturing, and to transmit such DNA to its progeny through at lea~t s~veral gQnerations. For eucaryotic cellular hosts, such stability i8 typically conferred by geno~ic integration of the PTH variant- ~ -encoding DNA. In ba~teria, which typically harbour transforminq DNA in the form of autonomously replicating lS pla~m?ds, such ~tability is usually ensured by culturing - -a strain carrying plasmid-conferre~ antibotic resistance in the presence of the antibiotic. ~ -For expression in the cellular host, DNA coding for a selected PTH variant ~ay be obtained using techniques that are well established in the art. For example, a DNA
sequence coding for a given PTH variant may be ~ -s synthesized de novo in accordance with methods standard ~ -in the gene synthesis art. Briefly, this entails the successive 3' to 5' coupling of suitably protected nucleotide reagents in an automated DNA synthesizer, and then the rscovery by gel purification of the deprotected polynucleotide. The block ligation approach may be employed, whereby "blocks" of oligonucleotide pairs, up ~-~
to about 80 nucleotides in length, are prepared and ligated in correct succession by overhang complementarity to assemble the variant-encoding DNA, as described~for example by Wosnick et al in Gene, 1989, 76:153. In an alternative approach, the desired DNA may be synthesized in toto, and then amplified by poly~erase chain reaction 35 (PCR), using the approach described by Barnett et al in t`'~
Nucl. Acids Res., 1990, 18~10):3094.
i y ~. .
It will be appreciated that alternative strategies ~
may also be applied to generate DNA coding for the -desired PTH variant. For instance, DNA coding for human PTH may be obtained and then used as a template e.g.
5 mutagenized site-specifically, to introduce the deslsed -amino acid change at the genetic level. DNA coding for human PTH may be obtained from an appropriate human cDNA
library, from a commercial source or by de novo synthesis according to t~e procedureR outlined above, and in accordance with the PTH-encoding nucleotide sequence reported for example by Hendy et al, Proc. Natl. Acad. ;
Sci. USA, 1981, 78:7365, incorporated herein by - reference, or a PTH-encoding ~quivalent thereof. The PTH-encoding DNA template may be converted to DNA coding ;~
for a PTH variant using the well established oligonucleotide-directed mutagenesis technique, as generally described for example by Xunkel et al, 1985, Proc. Natl. Acad. Sci. USA, 82:488. This technique is conv~niently accomplished with high efficiency using the E. coli-baRed system for synthesis and propogation of the altered gene in an appropriate vector, such as M13mpl8.
Kits useful Eor performing such procedures in vitro are available commercially. Also suitable for obtaining PTH ~ -`
variant-encoding DNA from a PTH-encoding template is the ~-2S related technigue in which site-directed mutagenesis is achieved using a PCR-based approach. One variant of this method, termed "recombinant PCR" is described by Higuchi et al, Nucl. Acids. Res., lg88, 16:7351, and a modified "megaprimer" PCR approach is described in Biotechniques, 1990, 8(1):404.
Once obtained, DNA coding for the desired PTH
variant is incorporated stably and expressibly into a ~-cellular host selected to serve in production of the PTH ~;-variant. A variety of organisms are suitable as hosts -for production of the PTH variants. ThesQ include eukaryotic hosts including yeasts such as Saccharomyces, Pichia and Kluveromyces, filamentous fungus hosts W093/20203 ~ PCT/CA93/00136 including AspergillUs species such as nidulans, niger (or awamori) and oryzae, insect cell hosts, and mammalian cell hosts including the CHo and COS cell lines. The PTH
variants are not dependent on glycosylation for activity, ;~
5 and thus can suitably be produced in bacterial hosts ~ ;
including Streptomyces, Bacillus and, preferably, in E.
coli. Recombinant DNA expression systems and culturing media/protocol~ enabling production in these hosts of a desired protein have already been established, and these 10 syste~s may be employed in the conventional ~anner for the specific purpose of producing PTH variants. E. coli production of PTH variants may be achieved, for examplQ, qv using expression syste~s ba~ed on the lac promoter (~ee Rabbani et al, Biochem., l990, 29:10080) and 15 expression/secretion systems based on the tac promoter (see Wong et al, EP 357,391). Yeast expre~sion may be achieved using expression systems based for example on the expre~sion controlling regions of the alpha-l mating ~ ~ ~
f~ctor gene a~ described by Gautvik et al in W088/03165.~ ~`
20 Production in Aspergillus may be achieved using secretion-~ ~
syste~s based on exprescion controlling region~ of the A. ~--nidulans alcA gene or the A. niger glucoa~ylase gene, as described for example by Gwynne et al in W086/06097. ~ ;
The P$H variant generated upon culturing of the ;~
production strain is extracted and purified using techniques that are al50 established in the art. In t general, the human PTH variants have characteristics that are similar generically to those exhibited by hu~an PTH, and may therefore be extracted and purified in 30 substantially the sa~e manner. Like PTH, the variants have a net positive charge at neutral pH (pI o~ about 9.3) and can be purified therefore by ion exchange chromatography, e.g. using cation exchange columns. The PTH variants are also, like PTH, hydrophobic in nature, 35 and may therefore be purified by hydrophobic interaction chro~tography e.g. on columns having a phenyl-Sepharose ~atrix. Al~o, of course, molecular sieves ~ay be u~ed to W093/20203 ' ~ PCT/CA93/00136 separate PTH variants from ot~er proteins unrelated by .
size, and affinity columns may be employed which comprise ;
PTH affinity agents such as hydroxyapatite or PTH
antibody. Preferably, purification of the PTH variant is S achieved by applying the protein mixture to a cation exchange column e.g. S-Sepharose, and then applying the eluted retentate to a column having a hydrophobic matrix e.g. a column havinq a phenyl, octyl or butyl side chain Quch a~ phenyl-Sepharose, phenyl-Superose, octyl-10 Sep~aro~e or butyl 650M. The retentate eluted ~rom the ;-hydrophobic matrix can then be subjected to final purification using reversed phase high performance liguid ~
chromatography (HPLC). ;
While the tryptic instability of human PTH typically lS demand~ that great care be taken during purification to guard against contamination by tryp~in in glassware and during handling, extraction and purification of the PTH
variants of the invention requires less stringent control mea~ure~. It is nevertheless desirable to exercise such control, in keeping with good laboratory and manufacturing practise.
For therapeutic use, a PTH variant is desirably purified to the extent that it migrates as a single peak on reversed phase HPLC, and exhibits a single band on polyacrylamide gel electrophoresis in the presence of SDS. Once purified, the PTH variant may be formulated to provide pharmaceutical composition~ ~uitable for treating the various clinical conditions for which PTH therapy is indicated. Compositions containing PTH variant are admini-~tered desirably to treat bone disordexs such as o~teoporosi~ and other osteopenic conditions, and for these purpo~es are suitably formulated either as injectables or ingestibles or for nasal insufflation, in accordance with established practi~e~ of protein drug formulation. Sterile injectable composition~ are particularly useful, and will generally compri~e an effective dose of the PTH variant, in admixutre with W093/20203 ~ '~ ~4 ~ PCT/CA93/00136 normal saline and suitable solubilizing agent e.g. dilute acetic acid. The PTH variant may alternatively be applied topically, as a cream, lotion, ointment or as an aerosol, to treat psoriasis and related skin disorders.
A suitable cream comprises an effective dose of the PTH
variant, in combination with carriers of standard composition e.g. in a triglyceride base.
A therapeutically effective dose of PTH variant, i.e. a do~e of PTH variant effective to treat a given clinical condition will depend of course on the nature and severity of the condition, and on such other factors as are normally considered and evaluated in clinical trials and by the attending physician. For treating osteoporosis, the PTH variant iB administred in amounts large enouqh to stimulate bone remodelling, but not so large as to cause net bone re~orption or sustained increase in serum calcium levels. Reference may be made to US patent 4,698,328 for guidance on the admini~tration of PTH to treat osteoporosis. Using the effective PTH
doses in a given clinical situation for guidance, the dose of PTH variant required to elicit a similar effect can be calculated based on the relative activity of the P~H variant. For example, ~His~]hPTH, [His~His~$eu~hPTH
and hPTH are substantially equipotent, and effective 25 doses of these PTH variants are thus similar to those of ;~
hPTH. It iB expected that the improved stability in the presence of trypsin of the PTH variants will provide for extended in vivo half-life, and thus somewhat smaller doses may be used or similar do es may be administered less frequently. It is anticipated that dosage sizes in the range from 0.05 ~g/kg to about l,000 ~g/kg, for exa~ple in the range from O.l ~g/kg to lO0 ~g/kg, and more suitably about 1-10 ~g/3cg will be clinically useful.
Like PTH, the PTH variants may be administered in 35 combination with other agents useful in treating a given --~
clinical condition. When treating osteoporosis and other -bone-related disorders for example, the PTH ~ariants may :
W093/20203 t,~ PCT/CA93/00136 be administered in conjunction with a dietary calcium ~ ~-supplement or with a vitamin D analogue (see US
4,698,328). Alternatively, the PTH variant may be administered~ preferably using a cyclic therapeutic ~;;
regimen, in combination with bisphosphonate~,as described - .... ,;
for example in US 4,761,406, or in combination with one or more bone therapeutic agent~ such as calcitonin and estrogen.
Exam~les ~-The example~ which follow describe production of huaan PTH and human PTH variants. Production of these proteins was achieved using, as a matter of convenience only, an E. coli-basQd system ~ubstantially as described by Wong and Sutherland in European patent application 89308?53.6 (publi~hed as EP357,391 on 7 March l990), the contents of which are incorporated herein by reference.
Thi~ sy~tem makes use of the commonly available E. coli JH101 strain as host and employs as vector a pUCl8 deriv~tive, designated pX. As is shown in Figure l, pX
incorporates the par element of pSClOl to enhance freguency of plasmid transmission, the lacIq gene of pMMB22 to enable overproduction of the lac repressor, and a PTH-excretion cassette. Incorporated in the excretion cassette is human PTH-encoding DNA that was synthesized using the block ligation technique reported by Wosnick et al, supr~, and in accordance with the PTH-encoding nucleotide sequance reported by Hendy et al, supr~.
Fused 5' of, and precisely to, the PTH-encoding DNA is the ~ignal ~equ~nce of the E. coli ompA gene, which is `?
capable of directing the PTH portion of the expression product across the host inner membrane, which migrates ultimately to the culturing medium. For regulated expression of the coding region, the plasmid operably -~
incorporates the tac promoter, the lac operator and a 3S con en~us ribo~omal binding site. Transcriptional ter~in~tion is controlled by the E. coli trpA gene terminator, and translational stop codons are provided in ;~
W093/~0203 ;~ 20 PCT/CA93/00136 all three reading frames, immediately 3' of the PTH~
encoding DNA.
Thus, the pX expression vector, used for the production of human PTH and PTH variants, is -5 substantially the same as that described by Wong and -~
Sutherland, supra, except that the multiple cloning site downstream of the PTH gene contains cleavage sites for - -~
the restriction enzymes ClaI, BamHI, XbaI, StuI and PstI, in the order indicated on Figure 1. The precise nucleotide seguence o~ the PTH-encoding region of the excretion cassette is illustrated in Figure 2.
Exam~le 1 - Production of human PTM(1-84) . ~
Plasmid pX was transfor~ed into competent E. coli JMlOi using standard procedures. Positive transformants were indentified following growth overnight at 30C on plates containing 2YT/agar and 70~g/ml ampicillin. PTH-producing transformants were then examined ~or PTH
activity, following growth in shake flasks, by IRMA
analysis of conditioned medium. and frozen stocks of the sQlected transformants were subsequently prepared by mixing an equal volume of the shake flask culture with sterile glycerol to yield 50%(v/v) glycerol stocks.
These stocks were subsequently stored at -80C. When needed, transformants were recovered from the frozen -stock by scraping, and were then streaked on ampicillin-containing plates of 2YT/agar.
To produce human PTH, freshly plated transformants were picked as single colonies and then inoculated into 50ml Erlenmeyer flasks containing 15ml of a liquid medium which contained 2YT, glucose and ampicillin in the standard mixture. Following overnight growth with ~-shaking at 30C, the cultures were diluted 20-fold with fresh medium, and then grown for three hours at 30-C with shaking. Expression of the PTH-encoding DNA was then de-repressed by addition of l.OmM IPTG. After growth for , ' ,''"
'- .,., ' - -.
... .
w093/20203 PCT/CA93/00136 four hours in the presence of IPTG, the culture was -cooled to 4C and centrifuged. The supernatant was then harvested and human PTH contained therein was recovered -and assayed for PTH activity. .4 S To obtain sufficient quantities of human PTH(1-84) and the PTH variants for purification and bioassay, larger volumes of conditioned ~edia werQ collect~d. In particular, freshly plated transfor~ants were picked as single colonies and then inoculated into 500ml flasks containing 200~1 of the ~edium described above.
Following overnight growth with shaking at 30-C, the cultures were inoculated into 2L bioreactor~ containing l.SL of the liquid medium, and then grown for S hours ~t 30-C with stirring. Expression of the PTH- or PTH
1S variant-encoding DNA was then induced by add~tion of 1.O~M IPTG. After growth for 3-4 hours in the presence of IPTG, the culture was cooled to 4-C and centrifuged.
The supernatant was then harvested, and the PTH or PTH
variant contained therein was purified in the manner described in Example 5.
The examples which follow describe production of PTH
variants. To obtain DNA coding for these variants, the in vitro site-directed mutagenesis technique described by Kunkel et al, supra, was applied. To perform this procedure there was first obtained plasmid RX which is an Ml3mpl8-based version of the excretion cassette on pX, carrying the PTH-encoding DNA as a promoterless NruI/XbaI
insert. Plasmid RX thus served as the template for conducting mutagenesis on the PTH-encoding DNA, in order to gQnerat~ DNA coding for a desired PTH variant. The particular mutagenesis strategy is described in the ~ -examples below.
Exa~Dle 2 - Production of a tHis~] variant of PTH
To provide DNA coding for a PTH variant in which Arg~ is replaced by histidine, plasmid RX was first W093/20203 '~J~ y 22 PCT/CA93/00136 recovered in single stranded form and about l~g thereof was incubated, at 85OC in Hin buffer, with about lOOng of a mutagenic oligonucleotide capable of hybridizing specifically to that region of the PTH gene containing -the Arg~ codon. The specific sequence of the oligonucleotide, designated Pl, is shown below where underlining indicates the codon change relative to the PTH-encoding template shown in Figure 2:
0 Pl oligos 5' CAGCTSCrSi~iC~GCCASSCrAC 3' t-~pl~t~: 3'....... CTOCAACAASCOCTOCCTAACATC........ 5~ ~ :
` ' After slow cooling, the annealed fragment was tre~ted with DNA polymer~e I (Xlenow) in the presence lS of all four dNTPs, for about 2 hour~ at 37C and then for 4 ho~rs at roo~ temperature, in order to form the full length double-stranded plas~id, d~signated pRXPl.
Co~pet~nt ho~t JMlOl was then tran~formed by pRXPl, and plaques were screened by restriction digest analysis and ~ ;
20 by DNA sequencing to select those carrying the desired ~
mutation. ~ ;
pRXPl is then digested with NruI and XbaI and the re~ulting small fragment is isolated by low melting point ~;
agarose. Plasmid pX is similarly digested, and the large 2S NruI/XbaI fragment is isolated. The relevant isolated fragments are then ligated, to form plasmid pXPl, which ;-~
carries DNA coding for [His~]hPTH. This was confirmed by ;
restriction digest analysis and DNA sequencing.
Competent E. coli JMlOl was transformed with pXPl - `~
and the transformants were then ~elected in accordance with the procedures outlined in Example l. Supernatant ~ ' containing the [His~PTH was then obtained for subsequent -...
purification by culturing the pXPl transformant, in the ~ -manner described by example l. ~--~
. . . ~ . .-. ~
.... .
.~':' :,',..
'~' '.' "'"' .: , , , - ,.~' -: . :
'J
Exam~le 3 - Production of a ~His2sHis26Leu~] variant of PTH
In a manner similar to that described in Example 2, there was obtained DNA coding for a human PTH variant in which the native sequence Arg25Lys2~Lysn is replaced by the sequence His~His26Leu~. In particular, single stranded pRX was incubated with an oligonucleotide having the sequence provided below, where underlining indicates the ., codon change relative to the native PTH-encoding template -shown in Figure 2:
P2 oligo 5 ' CATCCTGCACCACGTGCTGCACCCASTC~AC~C~ 3 ~ :
PTH t~pl~t- 3 ' . . .CSACCACC~CCAACA~TCCCTCCCTAACATCACA. . . 5 ~ ~ .
:
A double stranded plasmid carrying the desirQd codon replacements, designated p~XP2, is then cut with NruI/XbaI and the isolated small fragment is ligated with the large fragment of NruI/XbaI-digested pX. E. coli was then transformed by the resulting plasmid pXP2, and the transformant wa~ cultured in the manner outlined in Example l to yield supernatant containing -tHis~His~Leu~]hpTH.
Exam~le 4 - Production of additional PTH variants In the manner substantially as described above in example 3, supernatants containing additional PTH
variants are obtained by culturing E. coli transformants habouring PTH variant-encoding DNA. To generate the variant-encoding DNA, DNA coding for native hPTH (Example
2 ) i5 used as template and is incubated with a mutagenic oligonucleotide having the sequence noted below. For convenien~e herein, the annealing flanks of each oligonucleotide are not represented. Rather, the seguence of the mutagenic oligonucleotide in the Arg~Lys~Lysn region is represented and the codon alteration is identified by underlining. The resulting amino acid change is also identified:
27 26 2s S PTH amino acids ............. ..........Ly~Ly-Arg PTH t-mpl~t- 3' ........... CTACCACCTCCAACAATCCCTCCCTAACATCACA.... 5' ;
oligo -qu-nc- 5' ......... CATCCTGCACl-b~lo~-]CACCCATTCTACTCT.... 3' ICln~l from -CrTCTTCTC-; IA~nY] from -CTTCTS~-; lTrpY) from - ; :
[L~u~] from -CTTCSTCAC-; lI l-Y] from -CTTCTT9~-; IAl~l from - ..
CTTCTTACC-; .~
lV~ fro~ -CTTCTT~a~-; lProYl from -CTTCTTCGC-; IPh-~ from - : ::
lS IsyP] from -CTSCT ~ -; [Sor"] from -CTTCTT_5~-; lTh P l fro~ ~
:, "' :, tHi-~H~-~I from -CTTGSGGTC-s lHi~YCln~l from -CTTCTGGTC-IHi-~A n~l fro -CSr9r~Q~-; lHi-~Trp~l from -CTTCCAGTC-I~ ~ ul from -CSS ~ Q_; lHi~2sIl~#l fro~ -cT~
Cln~GlnY~ fro~ -CTTCTGCTC-; lA-nYA-n#¦ from -CSSÇ~5i~-s : -: Cln~Hl-~l fro~ -CS5ÇIÇS~Q-; lHi-YAl~#l from -CTSACC~TC~
~i-~V~l~l from -CTTAACCTC-; lHi-~Prol from -CT~ÇGCGTC- :.:.~.. -ICln~A-n#l from -CSS9~SIi-; [ClnYAl~l fro~ -CTTAGCCTG- .
25 [Cln~V~l~] from -CTTAACC~G-; lClnYL-u~l from -CTTCAGCTC- - .
ClnUIl-#l from -CTTÇa~ÇTG-; IGlnUPro~] from -CTTCGGCTC-A-n~Nl-#] from -CrI5a5ai}I-; [A~n~Cln~] from -CTTCSGCTT- :~ -[A-nVAla~l from -CTTAGCGTT-; tAsn~Val~] from -CTTAACGTT-~A~n~L-u~] from -CTSCAGGTT-; [Asn~Ile~ from -CTSGATGTT- :: .
tA-nVPr P l from -cSICGGGST-.
[Hi-~HirD] from -GTGCT$GTG-;IHi~Leunl from -CAGCTTGTG-;
[N~-~A-nD] from -g~CTSÇ C-;[Hi~ClnD] from -CSGCS$Ç~-5 lN~-V$rpDl from -CCACTT_~_-;IHis~Ilen] from -GATCTTÇTG-;
35 [Ni-~Al~D] from -~QCTTÇSC-;[Hie Valnl from -AACCTTG$C-s ~ -[H~Pron] from - _ ;[Gln~Hi8~] from -GTGCT$CIG-;
[Gln~Lnu~] from -CAGCST~TG-;[Cln~AsnD] from -~CTTÇTC-;
Cln~Clnnl from -CTGCTTCTC-;lGln~Srp~] from -CCACTTCTG-; ~ -~
ICln~ll n) fro~ -z35rr~ ;[Cln Ala~l from -~Ç_CTTCTC~
40 (cln~v~lnl fro~ QCSSCTC-;[Cln~Pro~] from -CGCCT$C$C-s :::
lA-n~H~-nl fro~ -GTGcl~z~-SlA-n~L~u~l from -Çlç~3r~it~-s ~:
(a-nUa-nnl fro~ -Ç~CSTg~-;lA-n'Cln~l from -C$GC1~EGE-;
la-nVTr ~ l from -CCAC~$g~-;lA-n~Ile-l from -a~CTTÇr~
27 26 2s S PTH amino acids ............. ..........Ly~Ly-Arg PTH t-mpl~t- 3' ........... CTACCACCTCCAACAATCCCTCCCTAACATCACA.... 5' ;
oligo -qu-nc- 5' ......... CATCCTGCACl-b~lo~-]CACCCATTCTACTCT.... 3' ICln~l from -CrTCTTCTC-; IA~nY] from -CTTCTS~-; lTrpY) from - ; :
[L~u~] from -CTTCSTCAC-; lI l-Y] from -CTTCTT9~-; IAl~l from - ..
CTTCTTACC-; .~
lV~ fro~ -CTTCTT~a~-; lProYl from -CTTCTTCGC-; IPh-~ from - : ::
lS IsyP] from -CTSCT ~ -; [Sor"] from -CTTCTT_5~-; lTh P l fro~ ~
:, "' :, tHi-~H~-~I from -CTTGSGGTC-s lHi~YCln~l from -CTTCTGGTC-IHi-~A n~l fro -CSr9r~Q~-; lHi-~Trp~l from -CTTCCAGTC-I~ ~ ul from -CSS ~ Q_; lHi~2sIl~#l fro~ -cT~
Cln~GlnY~ fro~ -CTTCTGCTC-; lA-nYA-n#¦ from -CSSÇ~5i~-s : -: Cln~Hl-~l fro~ -CS5ÇIÇS~Q-; lHi-YAl~#l from -CTSACC~TC~
~i-~V~l~l from -CTTAACCTC-; lHi-~Prol from -CT~ÇGCGTC- :.:.~.. -ICln~A-n#l from -CSS9~SIi-; [ClnYAl~l fro~ -CTTAGCCTG- .
25 [Cln~V~l~] from -CTTAACC~G-; lClnYL-u~l from -CTTCAGCTC- - .
ClnUIl-#l from -CTTÇa~ÇTG-; IGlnUPro~] from -CTTCGGCTC-A-n~Nl-#] from -CrI5a5ai}I-; [A~n~Cln~] from -CTTCSGCTT- :~ -[A-nVAla~l from -CTTAGCGTT-; tAsn~Val~] from -CTTAACGTT-~A~n~L-u~] from -CTSCAGGTT-; [Asn~Ile~ from -CTSGATGTT- :: .
tA-nVPr P l from -cSICGGGST-.
[Hi-~HirD] from -GTGCT$GTG-;IHi~Leunl from -CAGCTTGTG-;
[N~-~A-nD] from -g~CTSÇ C-;[Hi~ClnD] from -CSGCS$Ç~-5 lN~-V$rpDl from -CCACTT_~_-;IHis~Ilen] from -GATCTTÇTG-;
35 [Ni-~Al~D] from -~QCTTÇSC-;[Hie Valnl from -AACCTTG$C-s ~ -[H~Pron] from - _ ;[Gln~Hi8~] from -GTGCT$CIG-;
[Gln~Lnu~] from -CAGCST~TG-;[Cln~AsnD] from -~CTTÇTC-;
Cln~Clnnl from -CTGCTTCTC-;lGln~Srp~] from -CCACTTCTG-; ~ -~
ICln~ll n) fro~ -z35rr~ ;[Cln Ala~l from -~Ç_CTTCTC~
40 (cln~v~lnl fro~ QCSSCTC-;[Cln~Pro~] from -CGCCT$C$C-s :::
lA-n~H~-nl fro~ -GTGcl~z~-SlA-n~L~u~l from -Çlç~3r~it~-s ~:
(a-nUa-nnl fro~ -Ç~CSTg~-;lA-n'Cln~l from -C$GC1~EGE-;
la-nVTr ~ l from -CCAC~$g~-;lA-n~Ile-l from -a~CTTÇr~
3 ~ '$~ PCI/CA93/00136 A~n~Al~ from -~Q_CTTGTS-;[Aen~Val~] f rom -AACCTSGTT-;
A~n~Pro~ from -CGGCTTGTT-~ .
[His~His~ from -GTGGTGACG-;tHie~Leu~) from -CAGGTGACG-;
S lHie~A~n~1 from -GTTGTCACG-;[H~-~Cln~ from -C$GGTGACC-;
~Hi-~Trp~] from -CCAGTGACG-t[Hi~ n~ from -GATGTGACC-;
~Hi-~Ala~ from -AGCGTGACC-;~Hi~Val~ from -AACGTGACC-;
tH~-~Pron1 from -CGGGTGACC-;[GlnYHl-~1 from -GTGCTCACC-;
ClnYL u~l from -CACCTGArC-;~Cln~A-n~1 from -CTTCSGACG-;
0 tClnYClnn) fro~ -CTGCTCACG-~Cln~$rp~] from -CCACTCACC-S
~Cln~Il-~1 fro~ -CATCTGACC-;IClnYAl-n1 from -A CCTCACC-;
~ClnYV~ln1 from -AACCTGACG-;~Cln~Pron1 from -CGGCSCACC-; ~--tA-nYH$-n1 fro~ -GTCGTTACG-;IA-n~L u~1 from -CAGGTTACC~
IAJn~A-nn1 fro~ ACC-;~A-nYCln~1 from -CTGCTTACC-;
IA-n~Tr p 1 from -C Q CSTACC-;~A-n~Il~n] from -GATCTTACC-S
A-nYAl~nl fro~ -ACCCTTACC-;IA-n~V-l~1 from -AACCTTACC-; -~
A-nYPronl from -CGGGTTACC- ;~
IHi-~H~-~HL-D) fro~ -GTGCTCGTC-5~Hi-~Cln~Hi-n] from -GTGCTGGTC-S -~
tH~ A n~H~-n~ fro~ -CIC9 ~ C-;tHi-~Trp2~H~ from -GTGCCAGTC- s H~-YL-u~Hi-nl from -GTGCACGTC-;~Hi-~ Ni-n1 f~om -GTGCATGTC-S
H~-~L~u~L u~I fro~ -CAGCACGTG-;~Hi-~Gln~L un1 from -CACCTGGTC-s ICln~H$-~h~1 from -GTGGTGCSC-;[Cln~Gln~Hl-~] from -CTGCTGCTC-Analogues of the PTH variants of the invention can be generated in substantially the same manner, but using template DNA that codes for an analogue of PTH. To generate an analogue that incorporates replacement of ~e.thionine by leucine at position 8, for example, a template coding for ELeu8]hPTH is obtained using the site-directed ~utagenesis technique as described in co-pending application WO92/11286 published 9 July 1992 and incorporated herein by reference, and the ~Leu4]hPTH-encoding DNA i5 then used as template for a second round of site-directed mutagenesis in which an oligonucleotide capable of introducing a desired codon change in the Arg~Lys~Lys~ region is utilized. ~:
W093/20203 ~ t~ ~J PCT/CA93/00136 Example 5 - Purification and Evaluation of PTH and PTH -~
variants The conditioned medium collected from the transformants of Examples 1-4 was, in each case, adjusted to about pH 4 with glacial acetic a~id, and the solution waQ centrifuged. The supernatant was harvested and then passed through a column containing the cation exchange resin S-Sepharo~e FastFlow (Pharmacia, bed volume 50ml) pre-equilibrated with O.04M ammonium acetate (pH4.0~.
Resin-bound PTH or PTH variant was eluted by applying a concentration gradient of ammonium acetate as eluant from -~
O.04M - l.OM ammonium acetate (pH4.0). PTH or the PTH .
variant eluted from the resin at about 0.6M ammonium acet~te. Eluant fractions, containing PTH or the PTH
variant (a~ measured by the Allegro two-site IRMA
purchased form Joldan Diagnostics, California, catalogue t40-2170, or by absorbance at 280nm), were combined to provide PTH or variant at about 60-70~ purity. -Samples of greater purity were obtained by su~jecting the combined fractions to a chromatographic ;~
separation using the resin phenyl-Sepharose ~astFlow ~-(Pharmacia). More particularly, the pH of the combined - -~
S-Sepharose fractions was adjusted to pH 8 with SN NaOH.
This solution was then applied to a column containing phenyl-Sepharose (6ml bed voluMe), pre-e~uilibrated with -~ -the buffer (6 volumes of l.OM ammonium acetate (pH4.0) and 4 volumes o~ 40mM ammonium acetate (pH4.0), then adjusted to pH 8~0 with 5N NaOH). PTH or variant ad~orbed to the column was then eluted usin~ as eluant a concentration gradient of buffer to 0.6M ammonium acetate (pH8.0).
Fractions containing PTH activity (as measured by Allegro two-site IRMA or monitored by A*w) were combined and then de~alted by pass2ge through a cartridge containing reversed phase C-18-like resin e.g. Sep-Pak (Waters Inc.) or Amberchrom CG71 resin (Toso Haas) pre- ~ -' ~
W093/20203 i ~ PCT/CA93/00136 equilibrated with 0.1% TFA. The PTH or variant bound to the resin was eluted with 0.1% TFA/80% acetronitrile and desalted preparations were frozen in liquid nitrogen, ;~
lyophilized and stored at -20C.
Thawed or fresh samples of human PTH(1-84) and of PTH variant~ obtained as described above were then evaluated for biological activity using a UMR-106 based adenylate cyclase a~ay and the protocol as described by Rabbini et al, 1988, Endocrinology, 123:2709, which is incorporated herein by reterence. A~ noted, rat osteosarcoma cells of the UMR line are cti~ulated by PTH
to activate adenylate cyclase, an enzyme which catalyzes intracellular conversion of ATP to it~ cylic ~onophosphate analogue, cAMP. In thi~ a~say therefore, lS PTH activity iQ determined by assaying the for~ation of cA~P in PTH-sti~ulated UMR cells. The results of the a~says, expres~ed in ter~s of EC~ (concentration of PTH
or variant effective for half-maximal sti~ulation of adenylate cyclase activity), are pre~ented in the following table:
' '' :
~'; ~ " '', ~. .- .:
: .:
WOg3/20203 '' PCT/CA93/00136 28 '~
Table 1 ~,, -. .: ' '-COMPO~ND ECj~ ~DM) hPTH(1-84) 0 8 , -tHi~] 1 3 ~-tHis~His~Leu~] 1 3 ~Tyr~l 1 5 ,, tGln~l 1 7 , tAsnU~ ' 1 8 ' ';'' : ~- - '-lThr~l 2 9 -~ , tHi8~Glnn] - ,~
tHi~Gln~l 4 -', tHi8UGlnnl 4 , ' tHi~UHis~ 4 ',~ -;
_ .,.
tHisULeiu~ _ _ ,",;,,,~
tHis~Gln~His~] 21 ~'~',' tGlnUHis~His~l 30 , ;-- _ It is evident from these results that PTH variants altered in the trypsin-sensitive region are substantially equipotent with human PTH The effeet of amino aeid '' replaeement in this region on sensitivity to tr,ypsin was ', nex~ evaluated by comparing trypsin effeets on PTH
variants relative to a human PTH control, in the following manner To mea~ure the ~ensitivity of a eo~pound (native PTH
or variant~) to di~e~tion with try,psin, duplieate ,~
ineubation~ were stablished for eaeh eo~pound in either the pre~enee of tryp8in (nTrypsin Ineubation~) or the absenee of trypsin ("Moek Incubation") For example, a ~'' . .
W093/20203 ~ y PCT/CA93/00136 lyophilized aliquot of PTH (or variant) was solubilized for 20 minutes at room temperature in lO mM acetic acid at a final concentration of l mg/mL (So ~g PTH or variant plus So ~L lO mM acetic acid). Following complete 5 solubilization in acetic acid, the sample was diluted -with buffer (50 mM Tris.HCl pH 7.5, HTris buffer", to a final concentration of l ~g/20.82 ~L). A fresh trypsin stock solution (TPCX treated Type XIII trypsin (bovine pancreas) fro~ Sig~a Chemical Co.) was prepared for each sxperiment by solubilizing trypsin in Tri~ buf~er to a final concentration of lO ~g/mL (20 ~g trypsin plus 2 mL
Tris buffer). The activity of the trypsin stoc~ solution was established spectrophometrically at the beginning and the end of each experiment by ~onitoring its ability to cleave th~ synthetic ~ubstrate N-~-benzoyl-L-arginine ethyl ether (8AEE). The appearance of the cleavage product was monitored as a function of time at 253 nm. A
fresh stock solution of soybean trypsin inhibi~or (SBTI) was prepared in Tris buffer at a final concentration of lO ~g/mL. The ability of the SBTI stock solution to guench the activity of the trypsin ctock solution was verified by the addition of an equal weight of SBT~ to trypsin in the trypsin/BAEE mixture (i.e. 5 ~g trypsin in trypsin/BAEE solution plu8 5 ~g SBTI solution~.
Parallel trypsin and mock incubations were established as follows. Identical amounts of the PTH or variant sample in Tris buffer were added to each of two tubes (416.4 ~L of PTH or variant sample in Tris buffer at 1 ~g/20.82 ~L for a total of 20 ~g of PTH or variant in each of two tubes). The trypsin incubation received an amount of trypsin stock solution in Tris buffer so that the final weight of trypsin to PTH (or variant) was 1:500 (w:w) te.g. 0.4 ~g of trypsin was added to 20 ~g of PTH or variant or 4 ~L of trypsin stock solution -containing lO ~g/mL was added to 416.2 ~L of PIH or v~riant solution containing l ~g/20.82 ~L). The mock incubation recei-Jed Tris buffer alone lacking trypsin in W093/20203 !. '~ 9 PCT/CA93/~0136 an amount equal to the volume of trypsin used for the parallel incubation (e g 4 ~L of Tris buffer added to 416 2 ~L of PTH or variant solution containing l ~g/20 82 ~L) The parallel tubes were then incubated ~or 4 hours 5 at room temperature and were then "quenched" by the --addition of an amount of SBTI sufficient to inhibit completely the trypsin (4 ~L Or SSTI solution in Tris buffer at a concentration of lO ~g SBTI/mL was added to each tube) A portion of each of the incubation mixture~
wa~ used to construct a s-ries of dilutions of PTH (or variant) to test in the UMR-106 cell adenylate cyclase assay Particularly, the quenched incubations were diluted appropriat~ly with assay bu~fer to a final conc-ntration of 5 x lO '7 M (200 ~L of gu~nched sample lS described above containing PTH or variant at 5 x lO
was diluted lO-fold with 1800 ~L assay bu~fer) Appropriate serial dilutions (from 5 x lO '7 M to 3 x lO ~
M) were ~ade with assay buffer for the trypsin incubation ~ -Q~pl-~ and for the mock incubation samples Dose-respon~e curves were generated and the relevant EC~
values were calculated EC~ and EC~ correspond to ~ -the EC~ values for the trypsin and mock incubations, respectively The trypsin sensitivity (TS) for PTH (or a variant) was calculated as the ratio of EC~/EC~ for the sample The loss in bioactivity as a result of trypsin digestion at one or more lysine or arginine residues in the N-terminal portion of PTH (e g Lys13, Arg~, Arp, Lys26, Lys~) will result in an increase in EC~r~ and thu~ a TS value that is greater than one The average TS values for PTH and eight variants are su~marized in Table 2 The relative resistance (RR) of a variant compared to native PTH was calculated as the ratio of the trypsin sensitivity of PTH to that of the analogue (i e RRn~ = TS - /TS~) The greater the RR
35 value, the more resistant the bioactivity of the variant -is ~o trypsin degradation ~.,'.:.,-. ..
,,. '', ', .. ... ~ . . ... ..... .. .. . . .
W O 93/20203 ~ ~ PCT/CA93/00136 Table 2: Retention o~ Biological Activity o~ PTH and Variant~
Following Incubation with Tryp~in Compou~d Ssyp~ ~-ltl~lty R-l~tl~- ~ t~o-~T~) ~) ~PTHt1-84) SO ~n - 7) 1.0(n - 7) tGln~ 23 (n - 2) 8(n - 2) .
tTy~ ] 7 (n - 3) 9(n ~ 2) tHi~l 10 (n - 3) 12(n - 2) ,~
~Asn~] 1.5 (n - 3) 2S(n - 2) tThr~ 1.7 (n - 1) 39~n ~ 1) ~His~Gln~HisnJ o.9 (n ~ 3) 50(n ~ 2) ~ -~
tHis~His~Leu~ 0.9 (n ~ 3) 60(n - 2) _ _ ~.- -, ' [Gln~is~His~ o.g (n - 3) 60(n - 2) From the r~-ult~ in T~ble 2 it i~ clear th~t all :~
variants at po~ition 25 ~re sub~tantially resi~t~nt to degradation by trypsin compar~d with native PTH, nnd that ~:
vari~nts substitutsd at positions 25, 26 and 27 are completely r~sistant by this assay.
The stability of P~H and Yariants to degradAtion by tryp~in was al~o quantified by SDS poly~crylamide gel electrophoresis (SD5-PAGE). Incubations of PTN or variant with trypsin wsre established. ~or example, a sufficient aliquot o~ lyophilized PTH or variant w~s ~irst solubilized for 20 minutes at room temperatur~ with lOmM acetic acid (final concentration o~ 1 mg~rL) and then diluted about 20-~old with Tri~ burfer. About 10 ~g ~:.
of PTH ~or vari~nt) was removed for a preincubation, ::-~ti~- zero~ ti~- point, ~nd wa~ lyophilized. The incubation~ w re initi~ted by addition of an appropriate amount of freshly prepared trypsin solution in Tris buffer for a final trypsin to PTH (or variant) ratio of 1:200 (w/w). The samples were incubated at room -~-~
temperature. Aliguots of the appropriate volume S corresponding to an initial amount of undigested PTH (or variant) of 10 ~g were removed at specific time~ (2, 5 -and 20 minutes). These were quenched immediately by adding an equal weight of SBTI to trypsin (either 5 ~L of ~--;
SBTI in Tri~ at a concentration of 10 ~g/mL or 0.05 ~g 10 SBTI was added to the time point containing 0.05 ~g -`
trypsin and 10 ~g initial equivalent amount of PTH or variant in Tris buffer). Samples quenched at the various time poin~s were then lyophilized.
Analyses of the lyophilized preincubation sample and lS the ~yophilized time point samples were carried out using SDS-PAGE methods such a~ those describ~d by Lae~
1970, Nature (London) 227:680-685. An improved SDS-PAGE
system for the analysis of samples such a~ intact and frago-nted PTH with low to Joderate a unts of salt i5 described by Schagger and von Jagow (1987) Analytical Biochemistry 166: 368-379 and was used for the analysis of the trypsin digestion time course studies.
Lyophilized samples containing 10 ~g of intact and/or fragmented PTH or variant were solubilized in gel sample 25 buffer and subjected to electrophoresis using suitable ~--conditions to separate intact PTH from the smaller ~-fragments (16.5% acrylamide gel, BioRad Mini-Protean II -~-apparatus, 90 minutes, 100 mV). The gels were fixed and stained for protein with Coomassie Blue. The amount of intact, full length PTH present in each of the various samples (preincubation sample and time point Yamples) was --quantified using a gel scanning method, which measures - ~-the quantity of stain and therefore the amount of protein scanned in each band. Initially, a standard curve was - -35 constructed by loading various amounts of PTH in each --lane (1, 2, 4, 6, 8, 10 ~g per lane), scanning each lane, ~-~
and then const~ucting a standard curve of the quantity of w093/20203 ~ PCT/CA93/00136 stain at the mobility of PTH as a function of the amount of PTH loaded per lane. with the gel scanning system, the quantity of stain was proportional to the amount of PTH loaded in the range of l to lO ~g per lane. Using this standard curve, the amount of intact PTH pre~ent in each sample was calculated from the quantity of stain at the mobility of intact PTH.
The amount of PTH (or variant) in the preincubation sample was termed P0 (scanned amount corre~ponding to approximatQly 10 ~g intact PTH or variant) and the amount at ~ given ti~e, t, was defined as P, . A plot o~ P, / P0 v~r~us ti~ (t) for PTH showed the time-depend~nt loss in intact PTH upon incubation with trypsin. At a trypsin to PTH ratio of 1:200 (w/w) the tHis~His~Leu~] variant wa~ ~;
lS substantially resistant to proteolysis (see Table 3).
A~t~r 20 ~inutes, more than 50% of the initial ~ateri~
rerainod. Under thes~ conditions, the other Arg and Lys sit~s in the tHi~Hi~Leu~ variant are not very susceptibl~ to trypsin dige~tion. The half-life of PTH
(or v~riant) under these conditions of tryp~in degradation was measured from plots of P, / P0 ver~us time (t) as the time when 50% of the intact PTH (or variant) had been at least partially degraded (i.e. P, / P0 ~ 0.5).
The results in Table 3 indicate that variants with one or ~S two substitutions in region 25 - 27 have a longer half- ~ -lif~ compared with native PTH, and that variants with three substitutions are significantly more resistant to degradation. ~ -~
., .
W093/20203 i~ PCT/CA93/00136 Table 3: Half-life (minutes) from Tricine SDS-PAGE gel ~
analysis of PTH and variants at Trypsin:PTH = - -1:200 (w/w) 5 Co~poun~ Pt/Po v-r~u~
hPTH(1-84) 5 + 2 (n = 15) -~
. -~Gln~ 5 + 2 (n ~ 3) [Thr2~] 5 + 1 (n - 3) tHi~Hi~] 5 (n - 1) ~T~ 6 + 1 (n ~ 3) tNis~Glnn] 6 + 3 (n ~ 2) tABn~l 7 + 4 (n - 2) tHi~Gln~Hi~D~7 + 3 (n ~ 3) ,, tNi~] 8 ~ 5 (n - 3) ~
.- .: . .
tGln~Hi~Hi~n]15 + 4 (n - 3) -~ :',~, [His~His~Le~]~ 20 (n - 2) Pt/Po - Amount of PTH or variant remaining at time t -divided by amount of PTH or variant at time 0 ~
-Examvle 6 - In vivo efficacy study The tHis~His~Leu~ variant was also evaluated in vivo for its effect on skeletal tissue, in an ovariectomized rat model of osteoporosis. Formulations of this variant were first prepared by recon~tituting the variant from lyophilized powder in O.lM acetic acid, then diluting to lOmM acetic acid by addition of nor~al ~aline vehicle and 2% ~v/v) heat-inactivated rat ~eru~, to g-nerate a ~tock ~olution containing the variant at a concentration o~ about 150~g/ml. The for~ulation wa~
,c 1 ~
W O 93/20203 PC~r/CA93/00136 then injected sub-cutaneously at the nape of the neck, in ~,, , selected volumes representing dosage sizes of 25~g~kg and ;- ' 150~ug/kg (8 rats/ each dose) A third group of rats received human P~H at a dose of 150 ~g/kg and a fourth group received vehicle alone Treatment regimen consisted of single dose administration once daily for 28 days , ' After treatment, the rats were sacrificed and ; ,~
evaluated by total skeletal calciu~ scan Relative to ,,`
rats receiving vehicle alone, the whole body calcium "~'', lO Qcanning result~ indicated an i~provement in rats ,' receiving human PTH (5 8% increase), and a still further '~
improvement (9 6% increase) in rats receiving the ,~
tHi~His~Leun] variant This variant and native PTH were ~, equipotent in increasing the bone mineral dQnsity of the ' lS distal fe~ur and the fourth lumbar vertebrae It is ',~
b lieved that enhanc-d effects of this variant on total body calcium r~sult from its greater half-life in vivo ' ,,~'``'-i e it~ resi~tant to serum-borne trypsin and/or other ~',"',-enzy~es which otherwi~e degrade,and/or inactivate the -'-" ',",~
20 native hormone '' ,, ,, .~., .
A~n~Pro~ from -CGGCTTGTT-~ .
[His~His~ from -GTGGTGACG-;tHie~Leu~) from -CAGGTGACG-;
S lHie~A~n~1 from -GTTGTCACG-;[H~-~Cln~ from -C$GGTGACC-;
~Hi-~Trp~] from -CCAGTGACG-t[Hi~ n~ from -GATGTGACC-;
~Hi-~Ala~ from -AGCGTGACC-;~Hi~Val~ from -AACGTGACC-;
tH~-~Pron1 from -CGGGTGACC-;[GlnYHl-~1 from -GTGCTCACC-;
ClnYL u~l from -CACCTGArC-;~Cln~A-n~1 from -CTTCSGACG-;
0 tClnYClnn) fro~ -CTGCTCACG-~Cln~$rp~] from -CCACTCACC-S
~Cln~Il-~1 fro~ -CATCTGACC-;IClnYAl-n1 from -A CCTCACC-;
~ClnYV~ln1 from -AACCTGACG-;~Cln~Pron1 from -CGGCSCACC-; ~--tA-nYH$-n1 fro~ -GTCGTTACG-;IA-n~L u~1 from -CAGGTTACC~
IAJn~A-nn1 fro~ ACC-;~A-nYCln~1 from -CTGCTTACC-;
IA-n~Tr p 1 from -C Q CSTACC-;~A-n~Il~n] from -GATCTTACC-S
A-nYAl~nl fro~ -ACCCTTACC-;IA-n~V-l~1 from -AACCTTACC-; -~
A-nYPronl from -CGGGTTACC- ;~
IHi-~H~-~HL-D) fro~ -GTGCTCGTC-5~Hi-~Cln~Hi-n] from -GTGCTGGTC-S -~
tH~ A n~H~-n~ fro~ -CIC9 ~ C-;tHi-~Trp2~H~ from -GTGCCAGTC- s H~-YL-u~Hi-nl from -GTGCACGTC-;~Hi-~ Ni-n1 f~om -GTGCATGTC-S
H~-~L~u~L u~I fro~ -CAGCACGTG-;~Hi-~Gln~L un1 from -CACCTGGTC-s ICln~H$-~h~1 from -GTGGTGCSC-;[Cln~Gln~Hl-~] from -CTGCTGCTC-Analogues of the PTH variants of the invention can be generated in substantially the same manner, but using template DNA that codes for an analogue of PTH. To generate an analogue that incorporates replacement of ~e.thionine by leucine at position 8, for example, a template coding for ELeu8]hPTH is obtained using the site-directed ~utagenesis technique as described in co-pending application WO92/11286 published 9 July 1992 and incorporated herein by reference, and the ~Leu4]hPTH-encoding DNA i5 then used as template for a second round of site-directed mutagenesis in which an oligonucleotide capable of introducing a desired codon change in the Arg~Lys~Lys~ region is utilized. ~:
W093/20203 ~ t~ ~J PCT/CA93/00136 Example 5 - Purification and Evaluation of PTH and PTH -~
variants The conditioned medium collected from the transformants of Examples 1-4 was, in each case, adjusted to about pH 4 with glacial acetic a~id, and the solution waQ centrifuged. The supernatant was harvested and then passed through a column containing the cation exchange resin S-Sepharo~e FastFlow (Pharmacia, bed volume 50ml) pre-equilibrated with O.04M ammonium acetate (pH4.0~.
Resin-bound PTH or PTH variant was eluted by applying a concentration gradient of ammonium acetate as eluant from -~
O.04M - l.OM ammonium acetate (pH4.0). PTH or the PTH .
variant eluted from the resin at about 0.6M ammonium acet~te. Eluant fractions, containing PTH or the PTH
variant (a~ measured by the Allegro two-site IRMA
purchased form Joldan Diagnostics, California, catalogue t40-2170, or by absorbance at 280nm), were combined to provide PTH or variant at about 60-70~ purity. -Samples of greater purity were obtained by su~jecting the combined fractions to a chromatographic ;~
separation using the resin phenyl-Sepharose ~astFlow ~-(Pharmacia). More particularly, the pH of the combined - -~
S-Sepharose fractions was adjusted to pH 8 with SN NaOH.
This solution was then applied to a column containing phenyl-Sepharose (6ml bed voluMe), pre-e~uilibrated with -~ -the buffer (6 volumes of l.OM ammonium acetate (pH4.0) and 4 volumes o~ 40mM ammonium acetate (pH4.0), then adjusted to pH 8~0 with 5N NaOH). PTH or variant ad~orbed to the column was then eluted usin~ as eluant a concentration gradient of buffer to 0.6M ammonium acetate (pH8.0).
Fractions containing PTH activity (as measured by Allegro two-site IRMA or monitored by A*w) were combined and then de~alted by pass2ge through a cartridge containing reversed phase C-18-like resin e.g. Sep-Pak (Waters Inc.) or Amberchrom CG71 resin (Toso Haas) pre- ~ -' ~
W093/20203 i ~ PCT/CA93/00136 equilibrated with 0.1% TFA. The PTH or variant bound to the resin was eluted with 0.1% TFA/80% acetronitrile and desalted preparations were frozen in liquid nitrogen, ;~
lyophilized and stored at -20C.
Thawed or fresh samples of human PTH(1-84) and of PTH variant~ obtained as described above were then evaluated for biological activity using a UMR-106 based adenylate cyclase a~ay and the protocol as described by Rabbini et al, 1988, Endocrinology, 123:2709, which is incorporated herein by reterence. A~ noted, rat osteosarcoma cells of the UMR line are cti~ulated by PTH
to activate adenylate cyclase, an enzyme which catalyzes intracellular conversion of ATP to it~ cylic ~onophosphate analogue, cAMP. In thi~ a~say therefore, lS PTH activity iQ determined by assaying the for~ation of cA~P in PTH-sti~ulated UMR cells. The results of the a~says, expres~ed in ter~s of EC~ (concentration of PTH
or variant effective for half-maximal sti~ulation of adenylate cyclase activity), are pre~ented in the following table:
' '' :
~'; ~ " '', ~. .- .:
: .:
WOg3/20203 '' PCT/CA93/00136 28 '~
Table 1 ~,, -. .: ' '-COMPO~ND ECj~ ~DM) hPTH(1-84) 0 8 , -tHi~] 1 3 ~-tHis~His~Leu~] 1 3 ~Tyr~l 1 5 ,, tGln~l 1 7 , tAsnU~ ' 1 8 ' ';'' : ~- - '-lThr~l 2 9 -~ , tHi8~Glnn] - ,~
tHi~Gln~l 4 -', tHi8UGlnnl 4 , ' tHi~UHis~ 4 ',~ -;
_ .,.
tHisULeiu~ _ _ ,",;,,,~
tHis~Gln~His~] 21 ~'~',' tGlnUHis~His~l 30 , ;-- _ It is evident from these results that PTH variants altered in the trypsin-sensitive region are substantially equipotent with human PTH The effeet of amino aeid '' replaeement in this region on sensitivity to tr,ypsin was ', nex~ evaluated by comparing trypsin effeets on PTH
variants relative to a human PTH control, in the following manner To mea~ure the ~ensitivity of a eo~pound (native PTH
or variant~) to di~e~tion with try,psin, duplieate ,~
ineubation~ were stablished for eaeh eo~pound in either the pre~enee of tryp8in (nTrypsin Ineubation~) or the absenee of trypsin ("Moek Incubation") For example, a ~'' . .
W093/20203 ~ y PCT/CA93/00136 lyophilized aliquot of PTH (or variant) was solubilized for 20 minutes at room temperature in lO mM acetic acid at a final concentration of l mg/mL (So ~g PTH or variant plus So ~L lO mM acetic acid). Following complete 5 solubilization in acetic acid, the sample was diluted -with buffer (50 mM Tris.HCl pH 7.5, HTris buffer", to a final concentration of l ~g/20.82 ~L). A fresh trypsin stock solution (TPCX treated Type XIII trypsin (bovine pancreas) fro~ Sig~a Chemical Co.) was prepared for each sxperiment by solubilizing trypsin in Tri~ buf~er to a final concentration of lO ~g/mL (20 ~g trypsin plus 2 mL
Tris buffer). The activity of the trypsin stoc~ solution was established spectrophometrically at the beginning and the end of each experiment by ~onitoring its ability to cleave th~ synthetic ~ubstrate N-~-benzoyl-L-arginine ethyl ether (8AEE). The appearance of the cleavage product was monitored as a function of time at 253 nm. A
fresh stock solution of soybean trypsin inhibi~or (SBTI) was prepared in Tris buffer at a final concentration of lO ~g/mL. The ability of the SBTI stock solution to guench the activity of the trypsin ctock solution was verified by the addition of an equal weight of SBT~ to trypsin in the trypsin/BAEE mixture (i.e. 5 ~g trypsin in trypsin/BAEE solution plu8 5 ~g SBTI solution~.
Parallel trypsin and mock incubations were established as follows. Identical amounts of the PTH or variant sample in Tris buffer were added to each of two tubes (416.4 ~L of PTH or variant sample in Tris buffer at 1 ~g/20.82 ~L for a total of 20 ~g of PTH or variant in each of two tubes). The trypsin incubation received an amount of trypsin stock solution in Tris buffer so that the final weight of trypsin to PTH (or variant) was 1:500 (w:w) te.g. 0.4 ~g of trypsin was added to 20 ~g of PTH or variant or 4 ~L of trypsin stock solution -containing lO ~g/mL was added to 416.2 ~L of PIH or v~riant solution containing l ~g/20.82 ~L). The mock incubation recei-Jed Tris buffer alone lacking trypsin in W093/20203 !. '~ 9 PCT/CA93/~0136 an amount equal to the volume of trypsin used for the parallel incubation (e g 4 ~L of Tris buffer added to 416 2 ~L of PTH or variant solution containing l ~g/20 82 ~L) The parallel tubes were then incubated ~or 4 hours 5 at room temperature and were then "quenched" by the --addition of an amount of SBTI sufficient to inhibit completely the trypsin (4 ~L Or SSTI solution in Tris buffer at a concentration of lO ~g SBTI/mL was added to each tube) A portion of each of the incubation mixture~
wa~ used to construct a s-ries of dilutions of PTH (or variant) to test in the UMR-106 cell adenylate cyclase assay Particularly, the quenched incubations were diluted appropriat~ly with assay bu~fer to a final conc-ntration of 5 x lO '7 M (200 ~L of gu~nched sample lS described above containing PTH or variant at 5 x lO
was diluted lO-fold with 1800 ~L assay bu~fer) Appropriate serial dilutions (from 5 x lO '7 M to 3 x lO ~
M) were ~ade with assay buffer for the trypsin incubation ~ -Q~pl-~ and for the mock incubation samples Dose-respon~e curves were generated and the relevant EC~
values were calculated EC~ and EC~ correspond to ~ -the EC~ values for the trypsin and mock incubations, respectively The trypsin sensitivity (TS) for PTH (or a variant) was calculated as the ratio of EC~/EC~ for the sample The loss in bioactivity as a result of trypsin digestion at one or more lysine or arginine residues in the N-terminal portion of PTH (e g Lys13, Arg~, Arp, Lys26, Lys~) will result in an increase in EC~r~ and thu~ a TS value that is greater than one The average TS values for PTH and eight variants are su~marized in Table 2 The relative resistance (RR) of a variant compared to native PTH was calculated as the ratio of the trypsin sensitivity of PTH to that of the analogue (i e RRn~ = TS - /TS~) The greater the RR
35 value, the more resistant the bioactivity of the variant -is ~o trypsin degradation ~.,'.:.,-. ..
,,. '', ', .. ... ~ . . ... ..... .. .. . . .
W O 93/20203 ~ ~ PCT/CA93/00136 Table 2: Retention o~ Biological Activity o~ PTH and Variant~
Following Incubation with Tryp~in Compou~d Ssyp~ ~-ltl~lty R-l~tl~- ~ t~o-~T~) ~) ~PTHt1-84) SO ~n - 7) 1.0(n - 7) tGln~ 23 (n - 2) 8(n - 2) .
tTy~ ] 7 (n - 3) 9(n ~ 2) tHi~l 10 (n - 3) 12(n - 2) ,~
~Asn~] 1.5 (n - 3) 2S(n - 2) tThr~ 1.7 (n - 1) 39~n ~ 1) ~His~Gln~HisnJ o.9 (n ~ 3) 50(n ~ 2) ~ -~
tHis~His~Leu~ 0.9 (n ~ 3) 60(n - 2) _ _ ~.- -, ' [Gln~is~His~ o.g (n - 3) 60(n - 2) From the r~-ult~ in T~ble 2 it i~ clear th~t all :~
variants at po~ition 25 ~re sub~tantially resi~t~nt to degradation by trypsin compar~d with native PTH, nnd that ~:
vari~nts substitutsd at positions 25, 26 and 27 are completely r~sistant by this assay.
The stability of P~H and Yariants to degradAtion by tryp~in was al~o quantified by SDS poly~crylamide gel electrophoresis (SD5-PAGE). Incubations of PTN or variant with trypsin wsre established. ~or example, a sufficient aliquot o~ lyophilized PTH or variant w~s ~irst solubilized for 20 minutes at room temperatur~ with lOmM acetic acid (final concentration o~ 1 mg~rL) and then diluted about 20-~old with Tri~ burfer. About 10 ~g ~:.
of PTH ~or vari~nt) was removed for a preincubation, ::-~ti~- zero~ ti~- point, ~nd wa~ lyophilized. The incubation~ w re initi~ted by addition of an appropriate amount of freshly prepared trypsin solution in Tris buffer for a final trypsin to PTH (or variant) ratio of 1:200 (w/w). The samples were incubated at room -~-~
temperature. Aliguots of the appropriate volume S corresponding to an initial amount of undigested PTH (or variant) of 10 ~g were removed at specific time~ (2, 5 -and 20 minutes). These were quenched immediately by adding an equal weight of SBTI to trypsin (either 5 ~L of ~--;
SBTI in Tri~ at a concentration of 10 ~g/mL or 0.05 ~g 10 SBTI was added to the time point containing 0.05 ~g -`
trypsin and 10 ~g initial equivalent amount of PTH or variant in Tris buffer). Samples quenched at the various time poin~s were then lyophilized.
Analyses of the lyophilized preincubation sample and lS the ~yophilized time point samples were carried out using SDS-PAGE methods such a~ those describ~d by Lae~
1970, Nature (London) 227:680-685. An improved SDS-PAGE
system for the analysis of samples such a~ intact and frago-nted PTH with low to Joderate a unts of salt i5 described by Schagger and von Jagow (1987) Analytical Biochemistry 166: 368-379 and was used for the analysis of the trypsin digestion time course studies.
Lyophilized samples containing 10 ~g of intact and/or fragmented PTH or variant were solubilized in gel sample 25 buffer and subjected to electrophoresis using suitable ~--conditions to separate intact PTH from the smaller ~-fragments (16.5% acrylamide gel, BioRad Mini-Protean II -~-apparatus, 90 minutes, 100 mV). The gels were fixed and stained for protein with Coomassie Blue. The amount of intact, full length PTH present in each of the various samples (preincubation sample and time point Yamples) was --quantified using a gel scanning method, which measures - ~-the quantity of stain and therefore the amount of protein scanned in each band. Initially, a standard curve was - -35 constructed by loading various amounts of PTH in each --lane (1, 2, 4, 6, 8, 10 ~g per lane), scanning each lane, ~-~
and then const~ucting a standard curve of the quantity of w093/20203 ~ PCT/CA93/00136 stain at the mobility of PTH as a function of the amount of PTH loaded per lane. with the gel scanning system, the quantity of stain was proportional to the amount of PTH loaded in the range of l to lO ~g per lane. Using this standard curve, the amount of intact PTH pre~ent in each sample was calculated from the quantity of stain at the mobility of intact PTH.
The amount of PTH (or variant) in the preincubation sample was termed P0 (scanned amount corre~ponding to approximatQly 10 ~g intact PTH or variant) and the amount at ~ given ti~e, t, was defined as P, . A plot o~ P, / P0 v~r~us ti~ (t) for PTH showed the time-depend~nt loss in intact PTH upon incubation with trypsin. At a trypsin to PTH ratio of 1:200 (w/w) the tHis~His~Leu~] variant wa~ ~;
lS substantially resistant to proteolysis (see Table 3).
A~t~r 20 ~inutes, more than 50% of the initial ~ateri~
rerainod. Under thes~ conditions, the other Arg and Lys sit~s in the tHi~Hi~Leu~ variant are not very susceptibl~ to trypsin dige~tion. The half-life of PTH
(or v~riant) under these conditions of tryp~in degradation was measured from plots of P, / P0 ver~us time (t) as the time when 50% of the intact PTH (or variant) had been at least partially degraded (i.e. P, / P0 ~ 0.5).
The results in Table 3 indicate that variants with one or ~S two substitutions in region 25 - 27 have a longer half- ~ -lif~ compared with native PTH, and that variants with three substitutions are significantly more resistant to degradation. ~ -~
., .
W093/20203 i~ PCT/CA93/00136 Table 3: Half-life (minutes) from Tricine SDS-PAGE gel ~
analysis of PTH and variants at Trypsin:PTH = - -1:200 (w/w) 5 Co~poun~ Pt/Po v-r~u~
hPTH(1-84) 5 + 2 (n = 15) -~
. -~Gln~ 5 + 2 (n ~ 3) [Thr2~] 5 + 1 (n - 3) tHi~Hi~] 5 (n - 1) ~T~ 6 + 1 (n ~ 3) tNis~Glnn] 6 + 3 (n ~ 2) tABn~l 7 + 4 (n - 2) tHi~Gln~Hi~D~7 + 3 (n ~ 3) ,, tNi~] 8 ~ 5 (n - 3) ~
.- .: . .
tGln~Hi~Hi~n]15 + 4 (n - 3) -~ :',~, [His~His~Le~]~ 20 (n - 2) Pt/Po - Amount of PTH or variant remaining at time t -divided by amount of PTH or variant at time 0 ~
-Examvle 6 - In vivo efficacy study The tHis~His~Leu~ variant was also evaluated in vivo for its effect on skeletal tissue, in an ovariectomized rat model of osteoporosis. Formulations of this variant were first prepared by recon~tituting the variant from lyophilized powder in O.lM acetic acid, then diluting to lOmM acetic acid by addition of nor~al ~aline vehicle and 2% ~v/v) heat-inactivated rat ~eru~, to g-nerate a ~tock ~olution containing the variant at a concentration o~ about 150~g/ml. The for~ulation wa~
,c 1 ~
W O 93/20203 PC~r/CA93/00136 then injected sub-cutaneously at the nape of the neck, in ~,, , selected volumes representing dosage sizes of 25~g~kg and ;- ' 150~ug/kg (8 rats/ each dose) A third group of rats received human P~H at a dose of 150 ~g/kg and a fourth group received vehicle alone Treatment regimen consisted of single dose administration once daily for 28 days , ' After treatment, the rats were sacrificed and ; ,~
evaluated by total skeletal calciu~ scan Relative to ,,`
rats receiving vehicle alone, the whole body calcium "~'', lO Qcanning result~ indicated an i~provement in rats ,' receiving human PTH (5 8% increase), and a still further '~
improvement (9 6% increase) in rats receiving the ,~
tHi~His~Leun] variant This variant and native PTH were ~, equipotent in increasing the bone mineral dQnsity of the ' lS distal fe~ur and the fourth lumbar vertebrae It is ',~
b lieved that enhanc-d effects of this variant on total body calcium r~sult from its greater half-life in vivo ' ,,~'``'-i e it~ resi~tant to serum-borne trypsin and/or other ~',"',-enzy~es which otherwi~e degrade,and/or inactivate the -'-" ',",~
20 native hormone '' ,, ,, .~., .
Claims (46)
1. A stability-enhanced variant of a parathyroid hormone compound harbouring the region Arg25Lys26Lys27, in which said region is replaced in said variant by an amino acid sequence selected from:
(a) X25Lys26ys27;
(b) Arg25Y26Z27 (c) X25Y26Lys27;
(d) X25Lys26Z27; and (e) X25Y26Z27 wherein X, Y and Z are independently selected, trypsin-insensitive amino acids.
(a) X25Lys26ys27;
(b) Arg25Y26Z27 (c) X25Y26Lys27;
(d) X25Lys26Z27; and (e) X25Y26Z27 wherein X, Y and Z are independently selected, trypsin-insensitive amino acids.
2. A stability-enhanced variant according to claim 1, which is a variant of a parathyroid hormone compound selected from mature parathyroid hormone, a biologically active analogue of mature parathyroid hormone and a biologically active fragment of mature parathyroid hormone.
3. A stability-enhanced variant according to claim 2, wherein each of X, Y and Z are independently selected, genetically encoded amino acids.
4. A stability-enhanced variant according to claim 3, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, histidine and proline.
5. A stability-enhanced variant according to claim 3, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, asparagine, glutamine, histidine and proline.
6. A stability-enhanced variant according to claim 3, wherein X is histidine.
7. A stability-enhanced variant according to claim 3, wherein Y is histidine.
8. A stability-enhanced variant according to claim 3, wherein Z is leucine.
9. A stability-enhanced variant of a parathyroid hormone compound according to claim 1, wherein said region is replaced in said variant by the amino acid sequence X25Y26Z27 and X, Y and Z are independently selected, trypsin-insensitive amino acids.
10. A stability-enhanced variant according to claim 9, which is a variant of a parathyroid hormone compound selected from mature parathyroid hormone, a biologically active analogue of mature parathyroid hormone and a biologically active fragment of mature parathyroid hormone.
11. A stability-enhanced variant according to claim 10, wherein each of X, Y and Z are independently selected, genetically encoded amino acids.
12. A stability-enhanced variant according to claim 11, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, histidine and proline.
13. A stability-enhanced variant according to claim 11, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, asparagine, glutamine, histidine and proline.
14. A stability-enhanced variant according to claim 11, wherein X is histidine.
15. A stability-enhanced variant according to claim 11, wherein Y is histidine.
16. A stability-enhanced variant according to claim 11, wherein Z is leucine.
17. A stability-enhanced variant according to claim 1, which is a variant of a parathyroid hormone compound selected from mature human parathyroid hormone, a biologically active analogue of mature human parathyroid hormone and a biologically active fragment of mature human parathyroid hormone.
18. A stability-enhanced variant according to claim 17, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, histidine and proline.
19. A stability-enhanced variant according to claim 17, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, asparagine, glutamine, histidine and proline.
20. A stability-enhanced variant according to claim 17, wherein X is histidine.
21. A stability-enhanced variant according to claim 17, wherein Y is histidine.
22. A stability-enhanced variant according to claim 17, wherein Z is leucine.
23. A stability-enhanced variant according to claim 17, wherein said region is replaced in said variant by the amino acid sequence X25Y26Z27 and X, Y and Z are independently selected, trypsin-insensitive amino acids.
24. A stability-enhanced variant according to claim 23, wherein each of X, Y and Z are independently selected, genetically encoded amino acids.
25. A stability-enhanced variant according to claim 24, wherein each of X, Y and Z are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, histidine and proline.
26. A stability-enhanced variant according to claim 24, wherein each of X, Y and 2 are independently selected from among the group of genetically encoded amino acids consisting of alanine, valine, leucine, isoleucine, asparagine, glutamine, histidine and proline.
27. A stability-enhanced variant according to claim 24, wherein X is histidine.
28. A stability-enhanced variant according to claim 25, wherein Y is histidine.
29. A stability-enhanced variant according to claim 25, wherein Z is leucine.
30. A stability-enhanced variant according to claim 17, selected from [Gln25]hPTH, [Tyr25]hPTH, [Asn25]hPTH and [Thr25]hPTH
31. A stability-enhanced variant according to claim 17, selected from [His25Gln25hPTH, [His25Gln25]hPTH, [His25His26]hPTH, [His25Leu26hPTH and [His26Gln27hPTH.
32. A stability-enhanced variant according to claim 17, which is [His25His26Leu27]hPTH.
33. A stability-enhanced variant according to claim 32, which is a biologically active fragment of [His25His26Leu27]hPTH.
34. A stability-enhanced variant according to claim 32, which is a biologically active analogue of [His25His26Leu27]hPTH.
35. A pharmaceutical composition comprising a therapeutically effective amount of a PTH variant as defined in claim 1, and a pharmaceutically acceptable carrier.
36. A pharmaceutical composition comprising a therapeutically effective amount of a human PTH variant as defined in claim 9, and a pharmaceutically acceptable carrier.
37. A pharmaceutical composition comprising a therapeutically effective amount of a PTH variant as defined in claim 17, and a pharmaceutically acceptable carrier.
38. A pharmaceutical composition comprising a therapeutically effective amount of a PTH variant as defined in claim 23, and a pharmaceutically acceptable carrier.
39. A cellular host having incorporated stably and expressibly therein a DNA molecule which codes for a PTH
variant as defined in claim 3.
variant as defined in claim 3.
40. A cellular host having incorporated stably and expressibly therein a DNA molecule which codes for a PTH
variant as defined in claim 11.
variant as defined in claim 11.
41. A cellular host having incorporated stably and expressibly therein a DNA molecule which codes for a PTH
variant as defined in claim 18.
variant as defined in claim 18.
42. A cellular host having incorporated stably and expressibly therein a DNA molecule which codes for a PTH
variant as defined in claim 24.
variant as defined in claim 24.
43. A method for producing a PTH variant having improved stability in the presence of trypsin, which comprises the step of culturing a cellular host as defined in claim 39.
44. A method for producing a human PTH variant having improved stability in the presence of trypsin, which comprises the step of culturing a cellular host as defined in claim 40.
45. A method for producing a human PTH variant having improved stability in the presence of trypsin, which comprises the step of culturing a cellular host as defined in claim 41.
46. A method for producing a human PTH variant having improved stability in the presence of trypsin, which comprises the step of culturing a cellular host as defined in claim 42.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US863,014 | 1977-12-21 | ||
US07/863,014 US5382658A (en) | 1992-04-03 | 1992-04-03 | Stability-enhanced variants of parathyroid hormone |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2132949A1 true CA2132949A1 (en) | 1993-10-14 |
Family
ID=25340025
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002132949A Abandoned CA2132949A1 (en) | 1992-04-03 | 1993-03-31 | Stability-enhanced variants of parathyroid hormone |
Country Status (7)
Country | Link |
---|---|
US (1) | US5382658A (en) |
EP (1) | EP0633935A1 (en) |
AU (1) | AU3883993A (en) |
CA (1) | CA2132949A1 (en) |
IL (1) | IL105283A0 (en) |
WO (1) | WO1993020203A2 (en) |
ZA (1) | ZA932390B (en) |
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US5317010A (en) * | 1991-10-10 | 1994-05-31 | Peter K. T. Pang | Parathyroid hormone analogues substituted at AA 25, 26, 27, and use in osteoporosis treatment |
US5814603A (en) * | 1992-06-12 | 1998-09-29 | Affymax Technologies N.V. | Compounds with PTH activity |
US5589452A (en) * | 1992-07-14 | 1996-12-31 | Syntex (U.S.A.) Inc. | Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis |
US5821225A (en) * | 1992-07-14 | 1998-10-13 | Syntex (U.S.A.) Inc. | Method for the treatment of corticosteroid induced osteopenia comprising administration of modified PTH or PTHrp |
US5977070A (en) * | 1992-07-14 | 1999-11-02 | Piazza; Christin Teresa | Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis |
AU672790B2 (en) * | 1992-07-15 | 1996-10-17 | Novartis Ag | Variants of parathyroid hormone and its fragments |
IT1255723B (en) * | 1992-10-09 | 1995-11-13 | USE OF PARATORMONE, ITS BIOLOGICALLY ACTIVE FRAGMENTS AND RELATED PEPTIDES, FOR THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS USEFUL IN THE PREVENTION AND THERAPY OF ABORTION AND PRETERMONE BIRTH AND IN GENERAL FOR THE TREATMENT OF GESTATION | |
US6100389A (en) * | 1995-04-21 | 2000-08-08 | Human Genome Sciences, Inc. | Polynucleotides encoding a human chemotactic protein |
US6075124A (en) * | 1994-05-16 | 2000-06-13 | Human Genome Sciences, Inc. | Human chemotactin protein |
US6028169A (en) * | 1997-03-31 | 2000-02-22 | Human Genome Sciences, Inc. | Chemokine β-6 antagonists |
JP4486256B2 (en) * | 1998-10-22 | 2010-06-23 | ザ・ジェネラル・ホスピタル・コーポレイション | Biologically active peptides and peptide derivatives of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) |
US7820393B2 (en) * | 1999-01-14 | 2010-10-26 | Scantibodies Laboratory, Inc. | Methods, kits and antibodies for detecting parathyroid hormone |
US6689566B1 (en) | 1999-01-14 | 2004-02-10 | Scantibodies Laboratory, Inc. | Methods, kits, and antibodies for detecting parathyroid hormone |
US20080108086A1 (en) * | 1999-06-02 | 2008-05-08 | Cantor Thomas L | Parathyroid hormone antagonists and uses thereof |
WO2004028444A2 (en) * | 1999-06-02 | 2004-04-08 | Scantibodies Laboratory, Inc. | Parathyroid hormone antagonists and uses thereof |
AU7734800A (en) * | 1999-09-29 | 2001-04-30 | General Hospital Corporation, The | Polypeptide derivatives of parathyroid hormone (pth) |
WO2003009804A2 (en) * | 2001-07-23 | 2003-02-06 | The General Hospital Corporation | Conformationally constrained parathyroid hormone (pth) analogs |
EP1531855A4 (en) | 2002-01-10 | 2009-07-22 | Osteotrophin Llc | Treatment of bone disorders with skeletal anabolic drugs |
US20030152588A1 (en) * | 2002-01-14 | 2003-08-14 | Hsu-Shan Huang | Chinese traditional medicines for psoriasis |
US7906480B2 (en) * | 2002-05-23 | 2011-03-15 | Michael Holick | Use of a parathyroid hormone peptide analogs for the treatment of vaginal atrophy |
US20040067526A1 (en) * | 2002-10-03 | 2004-04-08 | Cantor Thomas L. | Methods for diagnosing and guiding treatment of bone turnover disease |
US7795220B2 (en) * | 2003-03-19 | 2010-09-14 | The General Hospital Corporation | Conformationally constrained parathyroid hormones with alpha-helix stabilizers |
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EP1653985A4 (en) | 2003-07-17 | 2009-08-05 | Gen Hospital Corp | Conformationally constrained parathyroid hormone (pth) analogs |
US7465703B1 (en) | 2003-10-01 | 2008-12-16 | Scantibodies Laboratory, Inc. | Methods and kits useful for guiding osteoporosis related therapy |
BRPI0509788A (en) * | 2004-05-13 | 2007-10-23 | Alza Corp | apparatus and method for transdermal delivery of parathyroid hormone agents |
US7541140B2 (en) * | 2004-06-03 | 2009-06-02 | Scantibodies Laboratory, Inc. | Assessing risk for kidney stones using parathyroid hormone agonist and antagonist |
JP2009545320A (en) * | 2006-08-04 | 2009-12-24 | ザ ジェネラル ホスピタル コーポレイション | Polypeptide derivative of parathyroid hormone (PTH) |
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US4423037A (en) * | 1982-05-13 | 1983-12-27 | The General Hospital Corporation | Inhibitors of peptide hormone action |
IL78342A (en) * | 1985-04-04 | 1991-06-10 | Gen Hospital Corp | Pharmaceutical composition for treatment of osteoporosis in humans comprising a parathyroid hormone or a fragment thereof |
ATE253119T1 (en) * | 1985-04-15 | 2003-11-15 | Dsm Ip Assets Bv | USE OF THE GLUCOAMYLASE PROMOTOR FROM APERGILLUS |
US4761406A (en) * | 1985-06-06 | 1988-08-02 | The Procter & Gamble Company | Regimen for treating osteoporosis |
JP2598801B2 (en) * | 1986-07-18 | 1997-04-09 | ザ ユニバーシティー オブ メルボルン | Malignant-PTHrP active protein in humoral hypercalcemia |
EP0383751B1 (en) * | 1986-10-22 | 1994-03-09 | GAUTVIK, Kaare M., Dr. | Production of human parathyroid hormone from microorganisms |
US4771124A (en) * | 1987-05-26 | 1988-09-13 | Merck & Co., Inc. | Parathyroid hormone antagonists with simplified synthetic methodology |
US4968669A (en) * | 1988-05-09 | 1990-11-06 | Merck & Co., Inc. | Parathyroid hormone antagonists |
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JPH03504201A (en) * | 1989-02-23 | 1991-09-19 | ゲゼルシャフト・フュア・ビオテクノロギッシェ・フォルシュンク・ミット・ベシュレンクテル・ハフツング (ゲー・ベー・エフ) | DNA sequences encoding PTH variants, PTH variants, expression vectors, bacterial hosts, compositions for use and treatment |
JPH0532696A (en) * | 1990-09-28 | 1993-02-09 | Takeda Chem Ind Ltd | Parathyroid hormone derivative |
US5317010A (en) * | 1991-10-10 | 1994-05-31 | Peter K. T. Pang | Parathyroid hormone analogues substituted at AA 25, 26, 27, and use in osteoporosis treatment |
US5434246A (en) * | 1992-03-19 | 1995-07-18 | Takeda Chemical Industries, Ltd. | Parathyroid hormone derivatives |
-
1992
- 1992-04-03 US US07/863,014 patent/US5382658A/en not_active Expired - Lifetime
-
1993
- 1993-03-31 AU AU38839/93A patent/AU3883993A/en not_active Abandoned
- 1993-03-31 EP EP93907711A patent/EP0633935A1/en not_active Withdrawn
- 1993-03-31 CA CA002132949A patent/CA2132949A1/en not_active Abandoned
- 1993-03-31 WO PCT/CA1993/000136 patent/WO1993020203A2/en not_active Application Discontinuation
- 1993-04-02 IL IL105283A patent/IL105283A0/en unknown
- 1993-04-02 ZA ZA932390A patent/ZA932390B/en unknown
Also Published As
Publication number | Publication date |
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AU3883993A (en) | 1993-11-08 |
WO1993020203A3 (en) | 1993-11-25 |
IL105283A0 (en) | 1993-08-18 |
ZA932390B (en) | 1994-01-14 |
WO1993020203A2 (en) | 1993-10-14 |
US5382658A (en) | 1995-01-17 |
EP0633935A1 (en) | 1995-01-18 |
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